Antimicrobial nanoemulsion compositions and methods

ABSTRACT

The present invention relates to compositions and methods for decreasing the infectivity, morbidity, and rate of mortality associated with a variety of pathogenic organisms and viruses. The present invention also relates to methods and compositions for decontaminating areas colonized or otherwise infected by pathogenic organisms and viruses. Moreover, the present invention relates to methods and compositions for decreasing the infectivity of pathogenic organisms in foodstuffs.

The following application is a Continuation-in-Part of U.S. applicationSer. No. 09/965,447, filed Sep. 27, 2001, which is aContinuation-in-Part of U.S. application Ser. No. 09/891,086, filed Jun.25, 2001, which is a Continuation-in-Part of U.S. application Ser. No.09/751,059, filed Dec. 29, 2000, which is a Continuation-in-part of09/561,111, filed Apr. 28, 2000, which is a Continuation-in-part of09/474,866, filed Dec. 30, 1999, each of which claims priority to U.S.provisional application No. 60/131,638, filed Apr. 28, 1999. Each ofthese applications in hereby incorporated herein by reference in theirentireties.

This invention was made in part during work partially supported by theU.S. government under DARPA grant No. MDA972-97-1-0007. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for decreasingthe infectivity, morbidity, and rate of mortality associated with avariety of pathogens. The present invention also relates to methods andcompositions for decontaminating areas, samples, solutions, andfoodstuffs colonized or otherwise infected by pathogens andmicroorganisms.

BACKGROUND OF THE INVENTION

Pathogens such as bacteria, fungi, viruses, and bacterial spores areresponsible for a plethora of human and animal ills, as well ascontamination of food and biological and environmental samples. Thefirst step in microbial infections of animals is generally attachment orcolonization of skin or mucus membranes, followed by subsequent invasionand dissemination of the infectious microbe. The portals of entry ofpathogenic bacteria are predominantly the skin and mucus membranes.

In particular, bacteria of the Bacillus genus form stable spores thatresist harsh conditions and extreme temperatures. Contamination offarmlands with B. anthracis leads to a fatal disease in domestic,agricultural, and wild animals (See e.g., Dragon and Rennie, Can. Vet.J. 36:295 [1995]). Human infection with this organism usually resultsfrom contact with infected animals or infected animal products (Seee.g., Welkos et al., Infect. Immun. 51:795 [1986]). Human clinicalsyndromes include a pulmonary form that has a rapid onset and isfrequently fatal. The gastrointestinal and cutaneous forms of anthrax,although less rapid, can result in fatalities unless treatedaggressively (See e.g., Franz et al., JAMA 278:399 [1997]; and Pile etal., Arch. Intem. Med. 158:429 [1998]). Bacillus anthracis infection inhumans is no longer common due to effective animal controls that includevaccines, antibiotics and appropriate disposal of infected livestock.However, animal anthrax infection still represents a significant problemdue to the difficulty in decontamination of land and farms. In addition,there is concern about human infection brought about by warfare and/orterrorist activities.

While an anthrax vaccine is available (See e.g., Ivins et al., Vaccine13:1779 [1995]) and can be used for the prevention of classic anthrax,genetic mixing of different strains of the organism can render thevaccine ineffective (See e.g., Mobley, Military Med. 160:547 [1995]).The potential consequences of the use of Anthrax spores as a biologicalweapon was demonstrated by the accidental release of Bacillus anthracisfrom a military microbiology laboratory in the former Soviet Union.Seventy-seven cases of human anthrax, including 66 deaths, wereattributed to the accident. Some anthrax infections occurred as far as 4kilometers from the laboratory (See e.g., Meselson et al., Science266:1202 [1994]). Genetic analysis of infected victims revealed thepresence of either multiple strains or a genetically altered B.anthracis (See e.g., Jackson et al., Proc. Nat. Acad. of Sci. U.S.A.95:1224 [1998]).

Additionally, other members of the Bacillus genus are also reported tobe etiological agents for many human diseases. Bacillus cereus is acommon pathogen. It is involved in food borne diseases due to theability of the spores to survive cooking procedures. It is alsoassociated with local sepsis and wound and systemic infection (See e.g.,Drobniewski, Clin. Micro. Rev. 6:324 [1993]). Many bacteria readilydevelop resistance to antibiotics. An organism infected with anantibiotic-resistant strain of bacteria faces serious and potentiallylife-threatening consequences.

Examples of bacteria that develop resistance include Staphylococcus thatoften cause fatal infections, Pneumococci that cause pneumonia andmeningitis; Salmonella and E. coli that cause diarrhea; and Enterococcithat cause blood-stream, surgical wound and urinary tract infections(See e.g., Berkelman et. al., J. Infcet. Dis. 170(2):272 [1994]).

Although an invaluable advance, antibiotic and antimicrobial therapysuffers from several problems, particularly when strains of variousbacteria appear that are resistant to antibiotics. In addition,disinfectants/biocides (e.g., sodium hypochlorite, formaldehyde andphenols) that are highly effective against Bacillus spores, are not wellsuited for decontamination of the environment, equipment, or casualties.This is due to toxicity that leads to tissue necrosis and severepulmonary injury following inhalation of volatile fumes. The corrosivenature of these compounds also renders them unsuitable fordecontamination of sensitive equipment (See e.g., Alasri et al., Can. J.Micro. 39:52 [1993]; Beauchamp et al., Crit. Rev. Tox. 22:143 [1992];Hess et al., Amer. J. dent. 4:51 [1991]; Lineaweaver et al., Arch. Surg.120:267 [1985]; Morgan, Tox. Path. 25:291 [1997]; and Russell, Clin.Micro. 3; 99 [1990]).

Influenza A virus is a common respirator pathogen that is widely used asa model system to test anti-viral agents in vitro (See e.g., Karaivanovaand Spiro, Biochem. J. 329:511 [1998]; Mammen et al., J. Med. Chem.38:4179 [1995]; and Huang et al., FEBS Letters 291:199 [1991]), and invivo (See e.g., Waghorn and Goa, Drugs 55:721 [1998]; Mendel et al.,Antimicrob. Agents Chemother. 42:640 [1998]; and Smith et al., J. med.Chem. 41:787 [1998]). The envelope glycoproteins, hemagglutinin (HA) andneuraminidase (NA), which determine the antigenic specificity of viralsubtypes, are able to readily mutate, allowing the virus to evadeneutralizing antibodies. Current anti-viral compounds and neuraminidaseinhibitors are minimally effective and viral resistance is common.

Clearly, antipathogenic compositions and methods that decrease theinfectivity, morbidity, and mortality associated with pathogenicexposure are needed. Such compositions and methods should preferably nothave the undesirable properties of promoting microbial resistance, or ofbeing toxic to the recipient.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for decreasingthe infectivity, morbidity, and rate of mortality associated with avariety of pathogens. The present invention also relates to methods andcompositions for decontaminating areas, samples, solutions, andfoodstuffs colonized or otherwise infected by pathogens andmicroorganisms. Certain embodiments of the present compositions arenontoxic and may be safely ingested by humans and other animals.Additionally, certain embodiments of the present invention arechemically stable and non-staining.

In some embodiments, the present invention provides compositions andmethods suitable for treating animals, including humans, exposed topathogens or the threat of pathogens. In some embodiments, the animal iscontacted with effective amounts of the compositions prior to exposureto pathogenic organisms. In other embodiments, the animal is contactedwith effective amounts of the compositions after exposure to pathogenicorganisms. Thus, the present invention contemplates both the preventionand treatment of microbiological infections.

In other embodiments, the present invention provides compositions andmethods suitable for decontaminating solutions and surfaces, includingorganic and inorganic samples that are exposed to pathogens or suspectedof containing pathogens. In still other embodiments of the presentinvention, the compositions are used as additives to prevent the growthof harmful or undesired microorganisms in biological and environmentalsamples.

In preferred embodiments, decreased pathogenic organism infectivity,morbidity, and mortality is accomplished by contacting the pathogenicorganism with an oil-in-water nanoemulsion comprising an oil phase, anaqueous phase, and at least one other component. In some preferredembodiments, the emulsion further comprises a solvent. In some preferredembodiments, the solvent comprises an organic phosphate solvent. Instill other embodiments, the organic phosphate-based solvent comprisesdialkyl phosphates or trialkyl phosphates (e.g., tributyl phosphate). Instill other preferred embodiments, the emulsion further comprises analcohol. In preferred embodiments that employ solvents, the solvent isprovided in the oil phase of the composition.

In some embodiments, the compositions of the present invention furthercomprise one or more surfactants or detergents. In some embodiments, itis contemplated that the surfactant is a non-anionic detergent. Inpreferred embodiments, the non-anionic detergent is a polysorbatesurfactant. In other embodiments, the non-anionic detergent is apolyoxyethylene ether. Surfactants that find use in the presentinvention include, but are not limited to surfactants such as the TWEEN,TRITON, and TYLOXAPOL families of compounds.

In certain other embodiments, the compositions of the present inventionfurther comprise one or more cationic halogen containing compounds,including but not limited to, cetylpyridinium chloride. In yet otherembodiments, the compositions of the present invention further compriseone or more compounds that promote or enhance the germination(“germination enhancers”) of certain microorganism, and in particularthe spore form of certain bacteria. Germination enhancers contemplatedfor formulation with the inventive compositions include, but are notlimited to, L-alanine, Inosine, CaCl₂, and NH₄Cl, and the like. In stillfurther embodiments, the compositions of the present invention furthercomprise one or more compounds that increase the interaction(“interaction enhancers”) of the composition with microorganisms (e.g.,chelating agents like ethylenediaminetetraacetic acid, orethylenebis(oxyethylenenitrilo)tetraacetic acid in a buffer).Additionally, in still other embodiments of the present invention, theformulations further comprise coloring or flavoring agents (e.g., dyesand peppermint oil).

In some embodiments, the composition further comprises an emulsifyingagent to aid in the formation of emulsions. Emulsifying agents includecompounds that aggregate at the oil/water interface to form a kind ofcontinuous membrane that prevents direct contact between two adjacentdroplets. Certain embodiments of the present invention featureoil-in-water emulsion compositions that may readily be diluted withwater to a desired concentration without impairing their anti-pathogenicproperties.

In addition to discrete oil droplets dispersed in an aqueous phase,oil-in-water emulsions can also contain other lipid structures, such assmall lipid vesicles (e.g., lipid spheres that often consist of severalsubstantially concentric lipid bilayers separated from each other bylayers of aqueous phase), micelles (e.g., amphiphilic molecules in smallclusters of 50-200 molecules arranged so that the polar head groups faceoutward toward the aqueous phase and the apolar tails are sequesteredinward away from the aqueous phase), or lamellar phases (lipiddispersions in which each particle consists of parallel amphiphilicbilayers separated by thin films of water).

These lipid structures are formed as a result of hydrophobic forces thatdrive apolar residues (e.g., long hydrocarbon chains) away from water.The above lipid preparations can generally be described as surfactantlipid preparations (SLPs). SLPs are minimally toxic to mucous membranesand are believed to be metabolized within the small intestine (See e.g.,Hamouda et al., J. Infect. Disease 180:1939 [1998]). SLPs arenon-corrosive to plastics and metals in contrast to disinfectants suchas bleach. As such, formulations of the present invention based on SLPsare contemplated to be particularly useful against bacteria, fungi,viruses and other pathogenic entities.

Certain embodiments of the present invention contemplate methods fordecreasing the infectivity of microorganisms (e.g., pathogenic agents)comprising contacting the pathogen with a composition comprising anoil-in-water emulsion. In some preferred embodiments, the emulsion is inthe form of an oil phase distributed in an aqueous phase with asurfactant, the oil phase includes an organic phosphate based solventand a carrier oil. In some embodiments, two or more distinct emulsionsare exposed to the pathogen. In preferred embodiments, the emulsions arefusigenic and/or lysogenic. In preferred embodiments, the oil phase usedin the method comprises a non-phosphate based solvent (e.g., analcohol).

In specific embodiments, the contacting is performed for a timesufficient to kill the pathogenic agent or to inhibit the growth of theagent. In other embodiments, the present invention provides a method ofdecontaminating an environmental surface harboring harmful or undesiredpathogens. In one such embodiment, the pathogenic agent is associatedwith an environmental surface and the method comprises contacting theenvironmental surface with an amount of the composition sufficient fordecontaminating the surface. While it may be so desired, decontaminationneed not result in total elimination of the pathogen. In someembodiments, the compositions and methods further comprise dyes, paints,and other marking and identification compounds to as to ensure that atreated surface has been sufficiently treated with the compositions ofthe present invention.

In certain embodiments, an animal is treated internally with acomposition of the present invention. In some preferred embodiments, thecontacting is via intradermal, subcutaneous, intramuscular orintraperitoneal injection. In other embodiments, the contacting is viaoral, nasal, buccal, rectal, vaginal or topical administration. When thepresent compositions are administered as pharmaceuticals, it iscontemplated that the compositions further comprise pharmaceuticallyacceptable adjutants, excipients, stabilizers, diluents, and the like.In still further embodiments, the present invention contemplatescompositions further comprising additional pharmaceutically acceptablebioactive molecules (e.g., antibodies, antibiotics, means for nucleicacid transfection, vitamins, minerals, co-factors, etc.).

In some preferred embodiments, the present invention provides acomposition comprising an oil-in-water emulsion, said oil-in-wateremulsion comprising a discontinuous oil phase distributed in an aqueousphase, a first component comprising an alcohol or glycerol, and a secondcomponent comprising a surfactant or a halogen-containing compound. Theaqueous phase can comprise any type of aqueous phase including, but notlimited to, water (e.g., diH₂O, distilled water, tap water) andsolutions (e.g., phosphate buffered saline solution). The oil phase cancomprise any type of oil including, but not limited to, plant oils(e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseedoil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil,safflower oil, and sunflower oil), animal oils (e.g., fish oil), flavoroil, water insoluble vitamins, mineral oil, and motor oil. In somepreferred embodiments, the oil phase comprises 30-90 vol % of theoil-in-water emulsion (i.e., constitutes 30-90% of the total volume ofthe final emulsion), more preferably 50-80%. While the present inventionin not limited by the nature of the alcohol component, in some preferredembodiments, the alcohol is ethanol or methanol. Furthermore, while thepresent invention is not limited by the nature of the surfactant, insome preferred embodiments, the surfactant is a polysorbate surfactant(e.g. TWEEN 20, TWEEN 40, TWEEN 60, and TWEEN 80), apheoxypolyethoxyethanol (e.g., TRITON X-100, X-301, X-165, X-102, andX-200, and TYLOXAPOL) or sodium dodecyl sulfate. Likewise, while thepresent invention is not limited by the nature of the halogen-containingcompound, in some preferred embodiments, the halogen-containing compoundcomprises a cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides,tetradecyltrimethylammonium halides, cetylpyridinium chloride,cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride,cetylpyridinium bromide, cetyltrimethylammonium bromide,cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide,dodecyltrimethylammonium bromide, or tetrad ecyltrimethylammoniumbromide.

The emulsions may further comprise third, fourth, fifth, etc.components. In some preferred embodiments, an additional component is asurfactant (e.g., a second surfactant), a germination enhancer, aphosphate based solvent (e.g., tributyl phosphate), a neutramingen,L-alanine, ammonium chloride, trypticase soy broth, yeast extract,L-ascorbic acid, lecithin, p-hyroxybenzoic acid methyl ester, sodiumthiosulate, sodium citrate, inosine, sodium hyroxide, dextrose, andpolyethylene glycol (e.g., PEG 200, PEG 2000, etc.).

The present invention also provides non-toxic, non-irritant, acomposition comprising an oil-in-water emulsion, said oil-in-wateremulsion comprising a quaternary ammonium compound, wherein saidoil-in-water emulsion is antimicrobial against bacteria, virus, fungi,and spores. In some preferred embodiments, the oil-in-water emulsion hasno detectable toxicity to plants or animals (e.g., to humans). In otherpreferred embodiments, the oil-in-water emulsion causes no detectableirritation to plants or animals (e.g., to humans). In some embodiments,the oil-in-water emulsion further comprises any of the componentsdescribed above. Quaternary ammonium compounds include, but are notlimited to, N-alkyldimethyl benzyl ammonium saccharinate,1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol; 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride;2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride; alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride; alkyl bis(2-hydroxyethyl) benzyl ammonium chloride; alkyldemethyl benzyl ammonium chloride; alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzylammonium chloride; alkyl dimethyl benzyl ammonium chloride (100% C 14);alkyl dimethyl benzyl ammonium chloride (100% C 16); alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12); alkyl dimethyl benzylammonium chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12); alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14);alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14); alkyl dimethyl benzylammonium chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18);alkyl dimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C118); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;myristalkonium chloride (and) Quat RNIUM 14;N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride; trimethoxysily propyldimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammoniumchloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyldimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethylbenzylammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

The present invention also provides methods of making each of theemulsions disclosed herein. For example, the present invention providesa method of making a oil-in-water emulsion comprising emulsifying amixture, said mixture comprising an oil, an aqueous solution, a firstcomponent comprising an alcohol or glycerol, and a second componentcomprising a surfactant or a halogen-containing compound.

The present invention further provides methods for protecting (e.g.,protecting from contamination of a microorganism) or decontaminating anarea (e.g., decontaminating an area by removing or reducing the numberof microorganisms in the area) comprising exposing the area to acomposition comprising an oil-in-water emulsion (e.g., any of theoil-in-water emulsions described herein). The method may be applied toany type of area. For example, in some embodiments, the area comprises asolid surface (e.g., a medical device), a solution, the surface of anorganism (e.g., an external or internal portion of a human), or a foodproduct.

The present invention also provides methods for modifying any of theemulsions described herein, comprising: providing the emulsion andadding or removing a component from the emulsion to produce a modifiedemulsion. In some embodiments, the method further comprises the step oftesting the modified emulsion in a biological assay (e.g., anantimicrobrial assay to determine the effectiveness of the emulsion atreducing the amount of microorganisms associated with a treated area).The present invention also contemplates methods of using such modifiedemulsion in commerce. For example, in some embodiments, the methodfurther comprises the step of advertising the sale of the modifiedemulsion and/or selling the modified emulsion.

The present invention also provides systems comprising a delivery system(e.g., a container, dispenser, packaging etc.) containing any of theoil-in-water emulsions described herein. The present invention furthercomprises a system comprising a material in contact with any of theoil-in-water emulsions described herein. The present invention is notlimited by the nature of the material in contact with the emulsion. Forexample, materials include, but are not limited to, medical devices,solutions, food products, cleaning products, motor oils, creams, andbiological materials (e.g., human tissues).

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects and embodiments of thepresent invention. The invention may be better understood by referenceto one or more of these figures in combination with the description ofspecific embodiments presented herein.

FIG. 1 illustrates the bactericidal efficacy of an emulsion of thepresent invention on B. cereus spores.

FIG. 2A-FIG. 2C illustrate bacterial smears showing the bactericidalefficacy of an emulsion of the present invention on B. cereus spores.

FIG. 3 illustrates the sporicidal activity of different dilutions of anemulsion of the present invention on different B. anthracis spores.

FIG. 4 illustrates a comparison of the sporicidal activity of anemulsion of the present invention and bleach over time.

FIG. 5 illustrates a comparison of the sporicidal activity of anemulsion of the present invention and bleach over time.

FIG. 6 illustrates the sporicidal activity of different dilutions of anemulsion of the present invention in media on different B. anthracisspores.

FIG. 7 illustrates the time course for the sporicidal activity of anemulsion of the present invention against B. anthracis from Del Rio,Tex.

FIG. 8 depicts an electron micrograph of E. coli (10,000×).

FIG. 9 depicts an electron micrograph of E. coli treated with BCTP(10,000×).

FIG. 10 depicts an electron micrograph of E. coli treated with W₈₀8P(10,000×).

FIG. 11 depicts an electron micrograph of Vibrio cholerae (25,000×).

FIG. 12 depicts an electron micrograph of Vibrio cholerae treated withW₈₀8P (25,000×).

FIG. 13 depicts an electron micrograph of Vibrio cholerae treated withBCTP (25,000×).

FIG. 14 depicts an electron micrograph of Vibrio cholerae treated withX₈W₆₀PC (25,000×).

FIG. 15 illustrates the effect of BCTP, W₈₀8P and X₈W₆₀PC on influenza Aactivity.

FIG. 16 illustrates the sporicidal activity of BCTP against 4 differentBacillus species compared to that of X₈W₆₀PC against 2 Bacillus species.BCTP showed a significant sporicidal activity after 4 hours of treatmentagainst Bacillus cereus, Bacillus circulans, and Bacillus megateriumspores, but not against Bacillus subtilis spores. X₈W₆₀PC, in 4 hours,showed more effective killing against B. cereus and also had asporicidal activity against B. subtilis which was resistant to BCTP.

FIG. 17 illustrates the time course of the nanoemulsion sporicidalactivity against Bacillus cereus. Incubation with BCTP diluted 1:100resulted in 95% killing in 4 hours. Incubation with X₈W₆₀PC diluted1:1000 resulted in 95% killing in only 30 minutes.

FIG. 18 depicts electron micrographs of Bacillus cereus spores pre- andpost-treatment with BCTP. Note, the uniform density in the cortex andthe well-defined spore coat before treatment with BCTP. Spores after 4hours of BCTP treatment show disruption in both the spore coat and thecortex with loss of core components.

FIG. 19 illustrates the effects of germination inhibition andstimulation on the sporicidal activity of BCTP diluted 1:100. BCTPsporicidal activity was delayed in the presence of 10 mM D-alanine(germination inhibition), and accelerated in the presence of 50 μML-alanine and 50 μM Inosine (germination stimulation).

FIG. 20A-FIG. 20F depict gross and histologic photographs of animalsinjected subcutaneously with different combinations of BCTP and B.cereus spores. FIG. 20A and FIG. 20B illustrate animals that wereinjected with BCTP alone at a dilution of 1:10. There was no grosstissue damage and histology showed no inflammation. FIG. 20C and FIG.20D illustrate animals that were injected with 4×10⁷ Bacillus cereusspores alone subcutaneously. A large necrotic area resulted with anaverage area of 1.68 cm². Histology of this area showed essentiallycomplete tissue necrosis of the epidermis and dermis includingsubcutaneous fat and muscle. FIG. 20E and FIG. 20F depict mice that wereinjected with 4×10⁷ Bacillus spores which had been immediately premixedwith the BCTP nanoemulsion at final dilution 1:10. These animals showedminimal skin lesions with average area 0.02 cm² (an approximate 98%reduction from those lesions resulting from an untreated infection withspores). Histology in FIG. 20F indicates some inflammation, however mostof the cellular structures in the epidermis and dermis were intact. Allhistopathology is shown at 4× magnification.

FIG. 21A-FIG. 21F depict gross and histological photographs of animalswith experimental wounds infected with Bacillus cereus spores. FIG. 21Aand FIG. 21B depict mice with experimental wounds that were infectedwith 2.5×10⁷ Bacillus cereus spores but not treated. Histologicalexamination of these wounds indicated extensive necrosis and a markedinflammatory response. FIG. 21C and FIG. 21D depict mice with woundsthat were infected with 2.5×10⁷ Bacillus cereus spores and irrigated 1hour later with saline. By 48 hours, there were large necrotic areassurrounding the wounds with an average area of 4.86 cm². In addition,80% of the animals in this group died as a result of the infection.Histology of these lesions indicated total necrosis of the dermis andsubdermis and large numbers of vegetative Bacillus organisms. FIG. 21Eand FIG. 21F depict mice with wounds that were infected with 2.5×10⁷Bacillus cereus spores and irrigated 1 hour later with a 1:10 dilutionof BCTP. There were small areas of necrosis adjacent to the wounds (0.06cm²) which was reduced 98% compared to animals receiving spores andsaline irrigation. In addition, only 20% of animals died from thesewounds. Histology of these lesions showed no evidence of vegetativeBacillus illustrates several particular embodiments the variousemulsions of the present invention.

FIG. 22 illustrates the inhibition of influenza A infection bysurfactant lipid preparations. FIG. 22A represents BCTP, W₈₀8P, SS, andNN; FIG. 22B: BCTP and SS. Virus was incubated with SLPs for 30 min. andsubsequently diluted and overlaid on cells. Inhibition of influenza Ainfection was measured using cellular ELISA. Each data point representsthe mean of three replicates+/−one standard error.

FIG. 23 illustrates the efficacy of BCTP as an anti-influenza agent ascompared to TRITON X-100. Influenza A virus was treated with BCTP,tri(n-butyl)phosphate/TRITON X-100/soybean oil (TTO), TRITONX-100/soybean oil (TO), and TRITON X-100 (T) alone for 30 min. Theconcentration of TRITON X-100 was the same in all preparations used fortreatment. Inhibition of influenza A infection was measured usingcellular ELISA. Each data point represents the mean of threereplicates+/−one standard error.

FIG. 24 shows that BCTP does not affect adenovirus infectivity.Adenoviral vector (AD.RSV ntlacZ) was treated with three dilutions ofBCTP for 30 min. and subsequently used for transfection of 293 cells.Five days later the 6-galactosidase assay was performed. Each data pointrepresents the mean of eight replicates+/one standard error.

FIG. 25 illustrates the structures of influenza A and adenovirus viewedwith electron microscopy. Viruses were either untreated or incubatedwith BCTP at 1:100 dilution for 15 and 60 min at room temperature andwere subjected to electron microscopy fixation procedure as described inthe Examples. FIG. 25A illustrates the influenza A virus untreated; FIG.25B illustrates influenza A virus incubated with BCTP for 15 min; FIG.25C illustrates the adenovirus untreated; and FIG. 25D illustrates theadenovirus incubated with BCTP for 60 min. For all imagesmagnification=200,000×. The bar represents 200 nm.

FIG. 26 illustrates the antibacterial properties of 1% and 10% BCTP. Thebactericidal effect (% killing) was calculated as:

$\frac{{{cfu}({initial})} - {{cfu}\left( {{post}\text{-}{treatment}} \right)}}{{cfu}({initial})} \times 100$

FIG. 27 illustrates the antiviral properties of 10% and 1% BCTP asassessed by plaque reduction assays.

FIG. 28 illustrates exemplary organisms that are target for theemulsions of the present invention.

FIG. 29 illustrates several particular embodiments of the variousemulsion compositions invention and certain uses for the emulsions.

FIG. 30 illustrates several particular embodiments of the variousemulsion compositions invention and certain uses for the emulsions.

FIG. 31 schematically depicts various generalized formulations and usesof certain embodiments of the present invention. FIG. 31A shows the logreduction of E. coli by various nanoemulsions of the present inventionfor 10%, 1% and 0.10% dilutions of the nanoemulsion. FIG. 31B shows logreduction of B. globigii spores by various nanoemulsions of the presentinvention for 10%, 1% and 0.10% dilutions of the nanoemulsion. FIG. 31Cshows log reduction of influenza A (pfu/ml) by various nanoemulsions ofthe present invention for 10%, 1% and 0.10% dilutions of thenanoemulsion.

FIG. 32 shows a graph of the log reduction of S. typhimurium treatedwith an emulsion of the present invention in the presence of EDTA at 40°C.

FIG. 33 shows a graph of the log reduction of S. typhimurium treatedwith an emulsion of the present invention in the presence of EDTA at 50°C.

FIG. 34 shows the lytic effect of an emulsion of the present inventioncompared to the lytic effect of its non-emulsified ingredients.

FIG. 35 shows the log reduction of Mycobacteria fortuitum by an emulsionof the present invention at room temperature and 37° C.

FIG. 36 shows data for the decontamination of a surface using anemulsion of the present invention.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein the term “microorganism” refers to microscopic organismsand taxonomically related macroscopic organisms within the categories ofalgae, bacteria, fingi (including lichens), protozoa, viruses, andsubviral agents. The term microorganism encompasses both those organismsthat are in and of themselves pathogenic to another organism (e.g.,animals, including humans, and plants) and those organisms that produceagents that are pathogenic to another organism, while the organismitself is not directly pathogenic or infective to the other organism. Asused herein the term “pathogen,” and grammatical equivalents, refers toan organism, including microorganisms, that causes disease in anotherorganism (e.g., animals and plants) by directly infecting the otherorganism, or by producing agents that causes disease in another organism(e.g., bacteria that produce pathogenic toxins and the like).

As used herein the term “disease” refers to a deviation from thecondition regarded as normal or average for members of a species, andwhich is detrimental to an affected individual under conditions that arenot inimical to the majority of individuals of that species (e.g.,diarrhea, nausea, fever, pain, and inflammation etc). A disease may becaused or result from contact by microorganisms and/or pathogens.

The terms “host” or “subject,” as used herein, refer to organisms to betreated by the compositions of the present invention. Such organismsinclude organisms that are exposed to, or suspected of being exposed to,one or more pathogens. Such organisms also include organisms to betreated so as to prevent undesired exposure to pathogens. Organismsinclude, but are not limited to animals (e.g., humans, domesticatedanimal species, wild animals) and plants.

As used herein, the term “inactivating,” and grammatical equivalents,means having the ability to kill, eliminate or reduce the capacity of apathogen to infect and/or cause a pathological responses in a host.

As used herein, the term “fusigenic” is intended to refer to an emulsionthat is capable of fusing with the membrane of a microbial agent (e.g.,a bacterium or bacterial spore). Specific examples of fusigenicemulsions include, but are not limited to, W₈₀8P described in U.S. Pat.Nos. 5,618,840; 5,547,677; and 5,549,901 and NP9 described in U.S. Pat.No. 5,700,679, each of which is herein incorporated by reference intheir entireties. NP9 is a branched poly(oxy-1,2ethaneolyl),alpha-(4-nonylphenal)-omega-hydroxy-surfactant. While notbeing limited to the following, NP9 and other surfactants that may beuseful in the present invention are described in Table 1 of U.S. Pat.No. 5,662,957, herein incorporated by reference in its entirety.

As used herein, the term “lysogenic” refers to an emulsion that iscapable of disrupting the membrane of a microbial agent (e.g., abacterium or bacterial spore). An exemplary lysogenic composition isBCTP. In preferred embodiments of the present invention, the presence ofboth a lysogenic and a fusigenic agent in the same composition producesan enhanced inactivating effect than either agent alone. Methods andcompositions using this improved antimicrobial composition are describedin detail herein.

The term “emulsion,” as used herein, includes classic oil-in-waterdispersions or droplets, as well as other lipid structures that can formas a result of hydrophobic forces that drive apolar residues (i.e., longhydrocarbon chains) away from water and drive polar head groups towardwater, when a water immiscible oily phase is mixed with an aqueousphase. These other lipid structures include, but are not limited to,unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles,and lamellar phases. Similarly, the term “nanoemulsion,” as used herein,refers to oil-in-water dispersions comprising small lipid structures.For example, in preferred embodiments, the nanoemulsion comprise an oilphase having droplets with a mean particle size of approximately 0.1 orless to 5 microns (e.g., 0.1 to 1.0). The terms “emulsion” and“nanoemulsion” are often used herein, interchangeably, to refer to thenanoemulsions of the present invention.

As used herein, the terms “contacted” and “exposed,” refers to bringingone or more of the compositions of the present invention into contactwith a pathogen or a sample to be protected against pathogens such thatthe compositions of the present invention may inactivate themicroorganism or pathogenic agents, if present. The present inventioncontemplates that the disclosed compositions are contacted to thepathogens or microbial agents in sufficient volumes and/orconcentrations to inactivate the pathogens or microbial agents.

The term “surfactant” refers to any molecule having both a polar headgroup, which energetically prefers solvation by water, and a hydrophobictail which is not well solvated by water. The term “cationic surfactant”refers to a surfactant with a cationic head group. The term “anionicsurfactant” refers to a surfactant with an anionic head group.

The terms “Hydrophile-Lipophile Balance Index Number” and “HLB IndexNumber” refer to an index for correlating the chemical structure ofsurfactant molecules with their surface activity. The HLB Index Numbermay be calculated by a variety of empirical formulas as described byMeyers, (Meyers, Surfactant Science and Technology, VCH Publishers Inc.,New York, pp. 231-245 [1992]), incorporated herein by reference. As usedherein, the HLB Index Number of a surfactant is the HLB Index Numberassigned to that surfactant in McCutcheon's Volume 1: Emulsifiers andDetergents North American Edition, 1996 (incorporated herein byreference). The HLB Index Number ranges from 0 to about 70 or more forcommercial surfactants. Hydrophilic surfactants with high solubility inwater and solubilizing properties are at the high end of the scale,while surfactants with low solubility in water which are goodsolubilizers of water in oils are at the low end of the scale.

As used herein, the term “germination enhancers” describe compounds thatact to enhance the germination of certain strains of bacteria (e.g.,L-amino acids [L-alanine], CaCl₂, Inosine, etc).

As used herein the term “interaction enhancers” describes compounds thatact to enhance the interaction of an emulsion with the cell wall of abacteria (e.g., a Gram negative bacteria). Contemplated interactionenhancers include but are not limited to chelating agents (e.g.,ethylenediaminetetraacetic acid [EDTA],ethylenebis(oxyethylenenitrilo)tetraacetic acid [EGTA], and the like)and certain biological agents (e.g., bovine serum albumin [BSA] and thelike).

The terms “buffer” or “buffering agents” refer to materials which whenadded to a solution, cause the solution to resist changes in pH.

The terms “reducing agent” and “electron donor” refer to a material thatdonates electrons to a second material to reduce the oxidation state ofone or more of the second material's atoms.

The term “monovalent salt” refers to any salt in which the metal (e.g.,Na, K, or Li) has a net 1+ charge in solution (i.e., one more protonthan electron).

The term “divalent salt” refers to any salt in which a metal (e.g., Mg,Ca, or Sr) has a net 2+ charge in solution.

The terms “chelator” or “chelating agent” refer to any materials havingmore than one atom with a lone pair of electrons that are available tobond to a metal ion.

The term “solution” refers to an aqueous or non-aqueous mixture.

As used herein, the term “therapeutic agent,” refers to compositionsthat decrease the infectivity, morbidity, or onset of mortality in ahost contacted by a pathogenic microorganism or that preventinfectivity, morbidity, or onset of mortality in a host contacted by apathogenic microorganism. Such agents may additionally comprisepharmaceutically acceptable compounds (e.g., adjutants, excipients,stabilizers, diluents, and the like). In some embodiments, thetherapeutic agents of the present invention are administered in the formof topical emulsions, injectable compositions, ingestable solutions, andthe like. When the route is topical, the form may be, for example, acream, ointment, salve or spray.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse allergic or immunological reactions whenadministered to a host (e.g., an animal or a human). Moreover, incertain embodiments, the compositions of the present invention may beformulated for horticultural or agricultural use. Such formulationsinclude dips, sprays, seed dressings, stem injections, sprays, andmists. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, wetting agents (e.g.,sodium lauryl sulfate), isotonic and absorption delaying agents,disintrigrants (e.g., potato starch or sodium starch glycolate), and thelike.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin andmucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells which line hollow organs orbody cavities).

As used herein, the term “topically active agents” refers tocompositions of the present invention that elicit pharmacologicalresponses at the site of application (contact) to a host.

As used herein, the term “systemically active drugs” is used broadly toindicate a substance or composition which will produce a pharmacologicalresponse at a site remote from the point of application or entry into asubject.

As used herein, the term “medical devices” includes any material ordevice that is used on, in, or through a patient's body in the course ofmedical treatment (e.g., for a disease or injury). Medical devicesinclude, but are not limited to, such items as medical implants, woundcare devices, drug delivery devices, and body cavity and personalprotection devices. The medical implants include, but are not limitedto, urinary catheters, intravascular catheters, dialysis shunts, wounddrain tubes, skin sutures, vascular grafts, implantable meshes,intraocular devices, heart valves, and the like. Wound care devicesinclude, but are not limited to, general wound dressings, biologic graftmaterials, tape closures and dressings, and surgical incise drapes. Drugdelivery devices include, but are not limited to, needles, drug deliveryskin patches, drug delivery mucosal patches and medical sponges. Bodycavity and personal protection devices, include, but are not limited to,tampons, sponges, surgical and examination gloves, and toothbrushes.Birth control devices include, but are not limited to, inter uterindevices (IUDs), diaphragms, and condoms.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants or undesired compounds from a sample or composition. Asused herein, the term “substantially purified” refers to the removal offrom about 70 to 90%, up to 100%, of the contaminants or undesiredcompounds from a sample or composition.

As used herein, the term “surface” is used in its broadest sense. In onesense, the term refers to the outermost boundaries of an organism orinanimate object (e.g., vehicles, buildings, and food processingequipment, etc.) that are capable of being contacted by the compositionsof the present invention (e.g., for animals: the skin, hair, and fur,etc., and for plants: the leaves, stems, flowering parts, and fruitingbodies, etc.). In another sense, the term also refers to the innermembranes and surfaces of animals and plants (e.g., for animals: thedigestive tract, vascular tissues, and the like, and for plants: thevascular tissues, etc.) capable of being contacted by compositions byany of a number of transdermal delivery routes (e.g., injection,ingestion, transdermal delivery, inhalation, and the like).

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to animal cells or tissues. In another sense, it ismeant to include a specimen or culture obtained from any source, such asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises compositions and methods for decreasingthe infectivity, morbidity, and rate of mortality associated with avariety of microbial and pathogenic organisms. The present inventionalso relates to methods and compositions for decontaminating areascolonized or otherwise infected by pathogenic organisms. Moreover, thepresent invention relates to methods and compositions for decreasing theinfectivity of pathogenic organisms in foodstuffs. In preferredembodiments, decreased pathogenic organism infectivity, morbidity, andmortality is accomplished by contacting the pathogenic organism with anoil-in-water composition comprising an aqueous phase, and oil phase, anat least one other compound. In some preferred embodiments, thecompositions of the present invention are non-toxic, non-irritant, andnon-corrosive, while possessing potency against a broad spectrum ofmicroorganisms, including bacteria, fungi, viruses, and spores. Certainillustrative embodiments of the present invention are described below.The present invention is not limited to these specific embodiments. Thedescription is provided in the following sections: I) ExemplaryCompositions; II) Exemplary Formulation Techniques; III) Properties andActivities; IV) Uses; and V) Specific Examples.

I. Exemplary Compositions

In preferred embodiments, the emulsions of the present inventioncomprise (i) an aqueous phase; (ii) an oil phase; and at least oneadditional compound. In some embodiments of the present invention, theseadditional compounds are admixed into either the aqueous or oil phasesof the composition. In other embodiments, these additional compounds areadmixed into a composition of previously emulsified oil and aqueousphases. In certain of these embodiments, one or more additionalcompounds are admixed into an existing emulsion composition immediatelyprior to its use. In other embodiments, one or more additional compoundsare admixed into an existing emulsion composition prior to thecompositions immediate use.

Additional compounds suitable for use in the compositions of the presentinvention include but are not limited to one or more organic, and moreparticularly, organic phosphate based solvents, surfactants anddetergents, cationic halogen containing compounds, germinationenhancers, interaction enhancers, food additives (e.g., flavorings,sweetners, bulking agents, and the like) and pharmaceutically acceptablecompounds. Certain exemplary embodiments of the various compoundscontemplated for use in the compositions of the present invention arepresented below.

A. Aqueous Phase

In certain preferred embodiments, the emulsion comprises about 5 to 60,preferably 10 to 40, more preferably 15 to 30, vol. % aqueous phase,based on the total volume of the emulsion, although higher and loweramounts are contemplated. In preferred embodiments, the aqueous phasecomprises water at a pH of about 4 to 10, preferably about 6 to 8. Whenthe emulsions of the present invention contain a germination enhancer,the pH is preferably 6 to 8. The water is preferably deionized(hereinafter “DiH₂O”) or distilled. In some embodiments the aqueousphase comprises phosphate buffered saline (PBS). In those embodiments ofthe present invention intended for consumption by, or contact to, ahost, the aqueous phase, and any additional compounds provided in theaqueous phase, may further be sterile and pyrogen free.

B. Oil Phase and Solvents

In certain preferred embodiments, the oil phase (e.g., carrier oil) ofthe emulsion of the present invention comprises 30-90, preferably 60-80,and more preferably 60-70, vol. % of oil, based on the total volume ofthe emulsion, although higher and lower amounts are contemplated.Suitable oils include, but are not limited to, soybean oil, avocado oil,flaxseed oil, coconut oil, cottonseed oil, squalene oil, olive oil,canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, pineoil (e.g., 15%), Olestra oil, fish oils, flavor oils, water insolublevitamins and mixtures thereof. In particularly preferred embodiments,soybean oil is used. Additional contemplated oils include motor oils,mineral oils, and butter. In preferred embodiments of the presentinvention, the oil phase is preferably distributed throughout theaqueous phase as droplets having a mean particle size in the range fromabout 1-2 microns, more preferably from 0.2 to 0.8, and most preferablyabout 0.8 microns. In other embodiments, the aqueous phase can bedistributed in the oil phase. In some preferred embodiments, very smalldroplet sizes are utilized (e.g., less than 0.5 microns) to producestable nanoemulsion compositions. It is contemplated that small dropletcomposition also provide clear solutions, which may find desired use incertain product types.

In some embodiments, the oil phase comprises 3-15, preferably 5-10 vol.% of an organic solvent, based on the total volume of the emulsion,although higher and lower amounts are contemplated. While the presentinvention is not limited to any particular mechanism, it is contemplatedthat the organic phosphate-based solvents employed in the emulsionsserve to remove or disrupt the lipids in the membranes of the pathogens.Thus, any solvent that removes the sterols or phospholipids in themicrobial membranes finds use in the emulsions of the present invention.Suitable organic solvents include, but are not limited to, organicphosphate based solvents or alcohols. In preferred embodiments, theorganic phosphate based solvents include, but are not limited to,dialkyl- and trialkyl phosphates (e.g., tri-n-butyl phosphate [TBP]) inany combination. A particularly preferred trialkyl phosphate in certainembodiments comprises tri-n-butyl phosphate, which is a plasticizer.Moreover, in a preferred embodiment, each alkyl group of the di- ortrialkyl phosphate has from one to ten or more carbon atoms, morepreferably two to eight carbon atoms. The present invention alsocontemplates that each alkyl group of the di- or trialkyl phosphate mayor may not be identical to one another. In certain embodiments, mixturesof different dialkyl and trialkyl phosphates can be employed. In thoseembodiments comprising one or more alcohols as solvents, such solventsinclude, but are not limited to, methanol, ethanol, propanol andoctanol. In a particularly preferred embodiment, the alcohol is ethanol.In those embodiments of the present invention intended for consumptionby, or contact to, a host, the oil phase, and any additional compoundsprovided in the oil phase, may further be sterile and pyrogen free.

C. Surfactants and Detergents

In some embodiments, the compositions of the present invention furthercomprise one or more surfactants or detergents (e.g., from about 3 to15%, and preferably about 10%, although higher and lower amounts arecontemplated). While the present invention is not limited to anyparticular mechanism, and an understanding of the mechanism is notrequired to practice the present invention, it is contemplated thatsurfactants, when present in the compositions, help to stabilize thecompositions. Both non-ionic (non-anionic) and ionic surfactants arecontemplated. Additionally, surfactants from the BRIJ family ofsurfactants find use in the compositions of the present invention. Thesurfactant can be provided in either the aqueous or the oil phase.Surfactants suitable for use with the emulsions include a variety ofanionic and nonionic surfactants, as well as other emulsifying compoundsthat are capable of promoting the formation of oil-in-water emulsions.In general, emulsifying compounds are relatively hydrophilic, and blendsof emulsifying compounds can be used to achieve the necessary qualities.In some formulations, nonionic surfactants have advantages over ionicemulsifiers in that they are substantially more compatible with a broadpH range and often form more stable emulsions than do ionic (e.g.,soap-type) emulsifiers. Thus, in certain preferred embodiments, thecompositions of the present invention comprises one or more non-ionicsurfactants such as a polysorbate surfactants (e.g., polyoxyethyleneethers), polysorbate detergents, pheoxypolyethoxyethanols, and the like.Examples of polysorbate detergents useful in the present inventioninclude, but are not limited to, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80,etc.

TWEEN 60 (polyoxyethylenesorbitan monostearate), together with TWEEN 20,TWEEN 40 and TWEEN 80, comprise polysorbates that are used asemulsifiers in a number of pharmaceutical compositions. In someembodiments of the present invention, these compounds are also used asco-components with adjuvants. TWEEN surfactants also appear to havevirucidal effects on lipid-enveloped viruses (See e.g., Eriksson et al.,Blood Coagulation and Fibtinolysis 5 (Suppl. 3):S37-S44 [1994]).

Examples of pheoxypolyethoxyethanols, and polymers thereof, useful inthe present invention include, but are not limited to, TRITON (e.g.,X-100, X-301, X-165, X-102, X-200), and TYLOXAPOL. TRITON X-100 is astrong non-ionic detergent and dispersing agent widely used to extractlipids and proteins from biological structures. It also has virucidaleffect against broad spectrum of enveloped viruses (See e.g., Maha andIgarashi, Southeast Asian J. Trop. Med. Pub. Health 28:718 [1997]; andPortocala et al., Virologie 27:261 [1976]). Due to this anti-viralactivity, it is employed to inactivate viral pathogens in fresh frozenhuman plasma (See e.g., Horowitz et al., Blood 79:826 [1992]).

In particularly preferred embodiments, the surfactants TRITON X-100(t-octylphenoxypolyethoxyethanol), and/or TYLOXAPOL are employed. Someother embodiments, employ spermicides (e.g., Nonoxynol-9). Additionalsurfactants and detergents useful in the compositions of the presentinvention may be ascertained from reference works (e.g., McCutheon'sVolume 1: Emulsions and Detergents—North American Edition, 2000).

In some embodiments, as shown in FIG. 28, compositions that comprise asurfactant and an organic solvent are useful for inactivating envelopedviruses and Gram positive bacteria.

D. Cationic Halogen Containing Compounds

In some embodiments, the compositions of the present invention furthercomprise a cationic halogen containing compound (e.g., from about 0.5 to1.0 wt. % or more, based on the total weight of the emulsion, althoughhigher and lower amounts are contemplated). In preferred embodiments,the cationic halogen-containing compound is preferably premixed with theoil phase; however, it should be understood that the cationichalogen-containing compound may be provided in combination with theemulsion composition in a distinct formulation. Suitable halogencontaining compounds may be selected, for example, from compoundscomprising chloride, fluoride, bromide and iodide ions. In preferredembodiments, suitable cationic halogen containing compounds include, butare not limited to, cetylpyridinium halides, cetyltrimethylammoniumhalides, cetyldimethylethylammonium halides, cetyldimethylbenzylammoniumhalides, cetyltributylphosphonium halides, dodecyltrimethylammoniumhalides, or tetradecyltrimethylammonium halides. In some particularembodiments, suitable cationic halogen containing compounds comprise,but are not limited to, cetylpyridinium chloride (CPC),cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride,cetylpyridinium bromide (CPB), cetyltrimethylammonium bromide (CTAB),cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide,dodecyltrimethylammonium bromide, and tetrad ecyltrimethylammoniumbromide. In particularly preferred embodiments, the cationic halogencontaining compound is CPC, although the compositions of the presentinvention are not limited to formulation with an particular cationiccontaining compound.

In some embodiments, addition of 1.0% wt. or more of a cationiccontaining compound to the emulsion compositions of the presentinvention provides a composition that is useful in inactivatingenveloped viruses, Gram positive bacteria, Gram negative bacteria andfungi.

E. Germination Enhancers

In other embodiments of the present invention, the compositions furthercomprise one or more germination enhancing compounds (e.g., from about 1mM to 15 mM, and more preferably from about 5 mM to 10 mM, althoughhigher and lower amounts are contemplated). In preferred embodiments,the germination enhancing compound is provided in the aqueous phaseprior to formation of the emulsion. The present invention contemplatesthat when germination enhancers are added to the disclosed compositionsthe sporicidal properties of the compositions are enhanced. The presentinvention further contemplates that such germination enhancers initiatesporicidal activity near neutral pH (between pH 6-8, and preferably 7).Such neutral pH emulsions can be obtained, for example, by diluting withphosphate buffer saline (PBS) or by preparations of neutral emulsions.The sporicidal activity of the compositions preferentially occurs whenthe spores initiate germination.

In specific embodiments, it has been demonstrated that the emulsions ofthe present invention have sporicidal activity. While the presentinvention is not limited to any particular mechanism, it is believedthat the fusigenic component of the emulsions acts to initiategermination and before reversion to the vegetative form is complete thelysogenic component of the emulsion acts to lyse the newly germinatingspore. These components of the emulsion thus act in concert to leave thespore susceptible to disruption by the emulsions. The addition ofgermination enhancer further facilitates the anti-sporicidal activity ofthe emulsions of the present invention, for example, by speeding up therate at which the sporicidal activity occurs.

Germination of bacterial endospores and fungal spores is associated withincreased metabolism and decreased resistance to heat and chemicalreactants. For germination to occur, the spore must sense that theenvironment is adequate to support vegetation and reproduction. Theamino acid L-alanine stimulates bacterial spore germination (See e.g.,Hills, J. Gen. Micro. 4:38 [1950]; and Halvorson and Church, BacteriolRev. 21:112 [1957]). L-alanine and L-proline have also been reported toinitiate fungal spore germination (Yanagita, Arch Mikrobiol 26:329[1957]). Simple α-amino acids, such as glycine and L-alanine, occupy acentral position in metabolism. Transamination or deamination of α-aminoacids yields the glycogenic or ketogenic carbohydrates and the nitrogenneeded for metabolism and growth. For example, transamination ordeamination of L-alanine yields pyruvate which is the end product ofglycolytic metabolism (Embden-Meyerhof-Parnas Pathway). Oxidation ofpyruvate by pyruvate dehydrogenase complex yields acetyl-CoA, NADH, H⁺,and CO₂. Acetyl-CoA is the initiator substrate for the tricarboxylicacid cycle (Kreb's Cycle) which in turns feeds the mitochondrialelectron transport chain. Acetyl-CoA is also the ultimate carbon sourcefor fatty acid synthesis as well as for sterol synthesis. Simple α-aminoacids can provide the nitrogen, CO₂, glycogenic and/or ketogenicequivalents required for germination and the metabolic activity thatfollows.

In certain embodiments, suitable germination enhancing agents of theinvention include, but are not limited to, α-amino acids comprisingglycine and the L-enantiomers of alanine, valine, leucine, isoleucine,serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl estersthereof. Additional information on the effects of amino acids ongermination may be found in U.S. Pat. No. 5,510,104, herein incorporatedby reference in its entirety. In some embodiments, a mixture of glucose,fructose, asparagine, sodium chloride (NaCl), ammonium chloride (NH₄Cl),calcium chloride (CaCl₂) and potassium chloride (KCl) also may be used.In particularly preferred embodiments of the present invention, theformulation comprises the germination enhancers L-alanine, CaCl₂,Inosine and NH₄Cl. In some embodiments, the compositions furthercomprise one or more common forms of growth media (e.g., trypticase soybroth, and the like) that additionally may or may not itself comprisegermination enhancers and buffers.

The above compounds are merely exemplary germination enhancers and it isunderstood that other known germination enhancers will find use in thecompositions of the present invention. A candidate germination enhancershould meet two criteria for inclusion in the compositions of thepresent invention: it should be capable of being associated with theemulsions of the present invention and it should increase the rate ofgermination of a target spore in the when incorporated in the emulsionsof the present invention. One skilled in the 1 art can determine whethera particular agent has the desired function of acting as an germinationenhancer by applying such an agent in combination with the compositionsof the present invention to a target and comparing the inactivation ofthe target when contacted by the admixture with inactivation of liketargets by the composition of the present invention without the agent.Any agent that increases germination, and thereby decrease or inhibitsthe growth of the organisms, is considered a suitable enhancer for usein the present invention.

In still other embodiments, addition of a germination enhancer (orgrowth medium) to a neutral emulsion composition produces a compositionthat is useful in treating bacterial spores in addition to envelopedviruses, Gram negative bacteria, and Gram positive bacteria.

In some embodiments, the present invention provides antimicrobialcompositions, including compositions that do not comprise emulsion ornanoemulsions, that comprise a germination enhancer. For example,germination enhancers may be added to any other material (e.g.,commercial disinfectants, solutions, etc.) to promote germination andincrease the ability of a composition to kill or neutralize spores ascompared to the activity of the composition in the absence of thegermination enhancer(s).

F. Interaction Enhancers

In still other embodiments, the compositions of the present inventioncomprise one or more compounds capable of increasing the interaction ofthe compositions (i.e., “interaction enhancer”) with target pathogens(e.g., the cell wall of Gram negative bacteria such as Vibrio,Salmonella, Shigella and Pseudomonas). In preferred embodiments, theinteraction enhancer is preferably premixed with the oil phase; however,in other embodiments the interaction enhancer is provided in combinationwith the compositions after emulsification. In certain preferredembodiments, the interaction enhancer is a chelating agent (e.g.,ethylenediaminetetraacetic acid [EDTA] orethylenebis(oxyethylenenitrilo)tetraacetic acid [EGTA] in a buffer[e.g., tris buffer]). It is understood that chelating agents are merelyexemplary interaction enhancing compounds. Indeed, other agents thatincrease the interaction of the compositions of the present inventionwith microbial agents and/or pathogens are contemplated. In particularlypreferred embodiments, the interaction enhancer is at a concentration ofabout 50 to about 250 μM, although higher and lower amounts arecontemplated. One skilled in the art will be able to determine whether aparticular agent has the desired function of acting as an interactionenhancer by applying such an agent in combination with the compositionsof the present invention to a target and comparing the inactivation ofthe target when contacted by the admixture with inactivation of liketargets by the composition of the present invention without the agent.Any agent that increases the interaction and thereby decrease orinhibits the growth of the bacteria in comparison to that parameter inits absence is considered an interaction enhancer.

In some embodiments, the addition of an interaction enhancer to thecompositions of the present invention produces a composition that isuseful in treating enveloped viruses, some Gram positive bacteria andsome Gram negative bacteria.

G. Other Components

In some embodiments of the present invention, the nanoemulsioncomposition comprise one or more additional components to provide adesired property or functionality to the nanoemulsions. These componentsmay be incorporated into the aqueous phase or the oil phase of thenanoemulsions and may be added prior to or following emulsification. Forexample, in some embodiments, the nanoemulsions further comprise phenols(e.g., triclosan, phenyl phenol), acidifying agents (e.g., citric acid[e.g., 1.5-6%], acetic acid, lemon juice), alkylating agents (e.g.,sodium hydroxide [e.g., 0.3%]), buffers (e.g., citrate buffer, acetatebuffer, and other buffers useful to maintain a specific pH), andhalogens (e.g., polyvinylpyrrolidone, sodium hypochlorite, hydrogenperoxide).

II. Exemplary Formulations

In section A), set forth below, the present invention describesexemplary techniques for making generic formulations of the disclosedcompositions. Additionally, the present invention recites a number ofspecific, although exemplary, formulation recipes in section B) setforth below.

A. Formulation Techniques

The pathogen inactivating oil-in-water emulsions of the presentinvention can be formed using classic emulsion forming techniques. Inbrief, the oil phase is mixed with the aqueous phase under relativelyhigh shear forces (e.g., using high hydraulic and mechanical forces) toobtain an oil-in-water nanoemulsion. The emulsion is formed by blendingthe oil phase with an aqueous phase on a volume-to-volume basis rangingfrom about 1:9 to 5:1, preferably about 5:1 to 3:1, most preferably 4:1,oil phase to aqueous phase. The oil and aqueous phases can be blendedusing any apparatus capable of producing shear forces sufficient to forman emulsion such as French Presses or high shear mixers (e.g. FDAapproved high shear mixers are available, for example, from Admix, Inc.,Manchester, N.H.). Methods of producing such emulsions are described inU.S. Pat. Nos. 5,103,497 and 4,895,452, herein incorporated by referencein their entireties.

In preferred embodiments, the compositions used in the methods of thepresent invention comprise droplets of an oily discontinuous phasedispersed in an aqueous continuous phase, such as water. In preferredembodiments, the compositions of the present invention are stable, anddo not decompose even after long storage periods (e.g., one or moreyears). Certain compositions of the present invention are non-toxic andsafe when swallowed, inhaled, or contacted to the skin of a host. Thisis in contrast to many chemical microbicides, which are known irritants.Additionally, in some embodiments, the compositions are also non-toxicto plants.

The compositions of the present invention can be produced in largequantities and are stable for many months at a broad range oftemperatures. Undiluted, they tend to have the texture of a semi-solidcream and can be applied topically by hand or mixed with water. Diluted,they tend to have a consistency and appearance similar to skim milk, andcan be sprayed to decontaminate surfaces or potentially interact withaerosolized spores before inhalation. These properties provide aflexibility that is useful for a broad range of antimicrobialapplications. Additionally, these properties make the compositions ofthe present invention particularly well suited to decontaminationapplications.

As stated above, at least a portion of the emulsion may be in the formof lipid structures including, but not limited to, unilamellar,multilamellar, and paucliamellar lipid vesicles, micelles, and lamellarphases.

Some embodiments of the present invention employ an oil phase containingethanol. For example, in some embodiments, the emulsions of the presentinvention contain (i) an aqueous phase and (ii) an oil phase containingethanol as the organic solvent and optionally a germination enhancer,and (iii) TYLOXAPOL as the surfactant (preferably 2-5%, more preferably3%). This formulation is highly efficacious against microbes and is alsonon-irritating and non-toxic to mammalian users (and can thus becontacted with mucosal membranes).

In some other embodiments, the emulsions of the present inventioncomprise a first emulsion emulsified within a second emulsion, wherein(a) the first emulsion comprises (i) an aqueous phase; and (ii) an oilphase comprising an oil and an organic solvent; and (iii) a surfactant;and (b) the second emulsion comprises (i) an aqueous phase; and (ii) anoil phase comprising an oil and a cationic containing compound; and(iii) a surfactant.

B. Exemplary Formulations

The following description provides a number of exemplary emulsionsincluding formulations for compositions BCTP and X₈W₆₀PC. BCTP comprisesa water-in oil nanoemulsion, in which the oil phase was made fromsoybean oil, tri-n-butyl phosphate, and TRITON X-100 in 80% water.X₈W₆₀PC comprises a mixture of equal volumes of BCTP with W₈₀8P. W₈₀8Pis a liposome-like compound made of glycerol monostearate, refined oy asterols (e.g., GENEROL sterols), TWEEN 60, soybean oil, a cationic ionhalogen-containing CPC and peppermint oil. The GENEROL family are agroup of a polyethoxylated soya sterols (Henkel Corporation, Ambler,Pa.). Emulsion formulations are given in Table 1 for certain embodimentsof the present invention. These particular formulations may be found inU.S. Pat. Nos. 5,700,679 (NN); 5,618,840; 5,549,901 (W₈₀8P); and5,547,677, herein incorporated by reference in their entireties. Certainother emulsion formulations are presented in FIG. 29. Moreover, FIG. 30schematically presents generalized formulations and uses of certainembodiments of the present invention.

The X₈W₆₀PC emulsion is manufactured by first making the W₈₀8P emulsionand BCTP emulsions separately. A mixture of these two emulsions is thenre-emulsified to produce a fresh emulsion composition termed X₈W₆₀PC.Methods of producing such emulsions are described in U.S. Pat. Nos.5,103,497 and 4,895,452 (herein incorporated by reference in theirentireties). These compounds have broad-spectrum antimicrobial activity,and are able to inactivate vegetative bacteria through membranedisruption.

TABLE 1 Water to Oil Phase Ratio Name Oil Phase Formula (Vol/Vol) BCTP 1vol. Tri(N-butyl)phosphate   4:1 1 vol. TRITON X-100 8 vol. Soybean oilNN 86.5 g Glycerol monooleate   3:1 60.1 ml Nonoxynol-9 24.2 g GENEROL122 3.27 g Cetylpyridinium chloride 554 g Soybean oil W₈₀8P 86.5 gGlycerol monooleate 3.2:1 21.2 g Polysorbate 60 24.2 g GENEROL 122 3.27g Cetylpyddinium chloride 4 ml Peppermint oil 554 g Soybean oil SS 86.5g Glycerol monooleate 3.2:1 21.2 g Polysorbate 60 (1% bismuth in water)24.2 g GENEROL 122 3.27 g Cetylpyridinium chloride 554 g Soybean oil

The compositions listed above are only exemplary and those of skill inthe art will be able to alter the amounts of the components to arrive ata nanoemulsion composition suitable for the purposes of the presentinvention. Those skilled in the art will understand that the ratio ofoil phase to water as well as the individual oil carrier, surfactant CPCand organic phosphate buffer, components of each composition may vary.

Although certain compositions comprising BCTP have a water to oil ratioof 4:1, it is understood that the BCTP may be formulated to have more orless of a water phase. For example, in some embodiments, there is 3, 4,5, 6, 7, 8, 9, 10, or more parts of the water phase to each part of theoil phase. The same holds true for the W₈₀8P formulation. Similarly, theratio of Tri(N-butyl)phosphate:TRITON X-100:soybean oil also may bevaried.

Although Table 1 lists specific amounts of glycerol monooleate,polysorbate 60, GENEROL 122, cetylpyridinium chloride, and carrier oilfor W₈₀8P, these are merely exemplary. An emulsion that has theproperties of W₈₀8P may be formulated that has different concentrationsof each of these components or indeed different components that willfulfill the same function. For example, the emulsion may have betweenabout 80 to about 100 g of glycerol monooleate in the initial oil phase.In other embodiments, the emulsion may have between about 15 to about 30g polysorbate 60 in the initial oil phase. In yet another embodiment thecomposition may comprise between about 20 to about 30 g of a GENEROLsterol, in the initial oil phase.

The nanoemulsions structure of the certain embodiments of the emulsionsof the present invention may play a role in their biocidal activity aswell as contributing to the non-toxicity of these emulsions. Forexample, the active component in BCTP, TRITON-X100 shows less biocidalactivity against virus at concentrations equivalent to 11% BCTP. Addingthe oil phase to the detergent and solvent markedly reduces the toxicityof these agents in tissue culture at the same concentrations. While notbeing bound to any theory (an understanding of the mechanism is notnecessary to practice the present invention, and the present inventionis not limited to any particular mechanism), it is suggested that thenanoemulsion enhances the interaction of its components with thepathogens thereby facilitating the inactivation of the pathogen andreducing the toxicity of the individual components. It should be notedthat when all the components of BCTP are combined in one composition butare not in a nanoemulsion structure, the mixture is not as effective asan antimicrobial as when the components are in a nanoemulsion structure.

Numerous additional embodiments presented in classes of formulationswith like compositions are presented below. The effect of a number ofthese compositions as antipathogenic materials is provided in FIG. 31.The following compositions recite various ratios and mixtures of activecomponents. One skilled in the art will appreciate that the belowrecited formulation are exemplary and that additional formulationscomprising similar percent ranges of the recited components are withinthe scope of the present invention.

In certain embodiments of the present invention, the inventiveformulation comprise from about 3 to 8 vol. % of TYLOXAPOL, about 8 vol.% of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 60to 70 vol. % oil (e.g., soybean oil), about 15 to 25 vol. % of aqueousphase (e.g., DiH₂O or PBS), and in some formulations less than about 1vol. % of 1N NaOH. Some of these embodiments comprise PBS. It iscontemplated that the addition of 1N NaOH and/or PBS in some of theseembodiments, allows the user to advantageously control the pH of theformulations, such that pH ranges from about 7.0 to about 9.0, and morepreferably from about 7.1 to 8.5 are achieved. For example, oneembodiment of the present invention comprises about 3 vol. % ofTYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 64vol. % of soybean oil, and about 24 vol. % of DiH₂O (designated hereinas Y3EC). Another similar embodiment comprises about 3.5 vol. % ofTYLOXAPOL, about 8 vol. % of ethanol, and about 1 vol. % of CPC, about64 vol. % of soybean oil, and about 23.5 vol. % of DiH₂O (designatedherein as Y3.5EC). Yet another embodiment comprises about 3 vol. % ofTYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.067vol. % of 1N NaOH, such that the pH of the formulation is about 7.1,about 64 vol. % of soybean oil, and about 23.93 vol. % of DiH₂O(designated herein as Y3EC pH 7.1). Still another embodiment comprisesabout 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. %of CPC, about 0.67 vol. % of 1N NaOH, such that the pH of theformulation is about 8.5, and about 64 vol. % of soybean oil, and about23.33 vol. % of DiH₂O (designated herein as Y3EC pH 8.5). Anothersimilar embodiment comprises 15==V about 4% TYLOXAPOL, about 8 vol. %ethanol, about 1% CPC, and about 64 vol. % of soybean oil, and about 23vol. % of DiH₂O (designated herein as Y4EC). In still another embodimentthe formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1vol. % of CPC, and about 64 vol. % of soybean oil, and about 19 vol. %of DiH₂O (designated herein as Y8EC). A further embodiment comprisesabout 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. %of CPC, about 64 vol. % of soybean oil, and about 19 vol. % of 1×PBS(designated herein as Y8EC PBS).

In some embodiments of the present invention, the inventive formulationscomprise about 8 vol. % of ethanol, and about 1 vol. % of CPC, and about64 vol. % of oil (e.g., soybean oil), and about 27 vol. % of aqueousphase (e.g., DiH₂O or PBS) (designated herein as EC).

In the present invention, some embodiments comprise from about 8 vol. %of sodium dodecyl sulfate (SDS), about 8 vol. % of tributyl phosphate(TBP), and about 64 vol. % of oil (e.g., soybean oil), and about 20 vol.% of aqueous phase (e.g., DiH₂O or PBS) (designated herein as S8P).

In certain embodiments of the present invention, the inventiveformulation comprise from about 1 to 2 vol. % of TRITON X-100, fromabout 1 to 2 vol. % of TYLOXAPOL, from about 7 to 8 vol. % of ethanol,about 1 vol. % of cetylpyridinium chloride (CPC), about 64 to 57.6 vol.% of oil (e.g., soybean oil), and about 23 vol. % of aqueous phase(e.g., DiH₂O or PBS). Additionally, some of these formulations furthercomprise about 5 mM of L-alanine/Inosine, and about 10 mM ammoniumchloride. Some of these formulations comprise PBS. It is contemplatedthat the addition of PBS in some of these embodiments, allows the userto advantageously control the pH of the formulations. For example, oneembodiment of the present invention comprises about 2 vol. % of TRITONX-100, about 2 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1vol. % CPC, about 64 vol. % of soybean oil, and about 23 vol. % ofaqueous phase DiH₂O. In another embodiment the formulation comprisesabout 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about7.2 vol. % of ethanol, about 0.9 vol. % of CPC, about 5 mML-alanine/Inosine, and about 10 mM ammonium chloride, about 57.6 vol. %of soybean oil, and the remainder of 1×PBS (designated herein as 90%X2Y2EC/GE).

In alternative embodiments of the present invention, the formulationscomprise from about 5 vol. % of TWEEN 80, from about 8 vol. % ofethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g.,soybean oil), and about 22 vol. % of DiH₂O (designated herein asW₈₀5EC).

In still other embodiments of the present invention, the formulationscomprise from about 5 vol. % of TWEEN 20, from about 8 vol. % ofethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g.,soybean oil), and about 22 vol. % of DiH₂O (designated herein asW₈₀5EC).

In still other embodiments of the present invention, the formulationscomprise from about 2 to 8 vol. % of TRITON X-100, about 8 vol. % ofethanol, about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g.,soybean, or olive oil), and about 15 to 25 vol. % of aqueous phase(e.g., DiH₂O or PBS). For example, the present invention contemplatesformulations comprising about 2 vol. % of TRITON X-100, about 8 vol. %of ethanol, about 64 vol. % of soybean oil, and about 26 vol. % of DiH₂O(designated herein as X2E). In other similar embodiments, theformulations comprise about 3 vol. % of TRITON X-100, about 8 vol. % ofethanol, about 64 vol. % of soybean oil, and about 25 vol. % of DiH₂O(designated herein as X3E). In still further embodiments, theformulations comprise about 4 vol. % Triton of X-100, about 8 vol. % ofethanol, about 64 vol. % of soybean oil, and about 24 vol. % of DiH₂O(designated herein as X4E). In yet other embodiments, the formulationscomprise about 5 vol. % of TRITON X-100, about 8 vol. % of ethanol,about 64 vol. % of soybean oil, and about 23 vol. % of DiH₂O (designatedherein as X5E). Another embodiment of the present invention comprisesabout 6 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol.% of soybean oil, and about 22 vol. % of DiH₂O (designated herein asX6E). In still further embodiments of the present invention, theformulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % ofethanol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH₂O(designated herein as X8E). In still further embodiments of the presentinvention, the formulations comprise about 8 vol. % of TRITON X-100,about 8 vol. % of ethanol, about 64 vol. % of olive oil, and about 20vol. % of DiH₂O (designated herein as X8E O). In yet another embodimentcomprises 8 vol. % of TRITON X-100, about 8 vol. % ethanol, about 1 vol.% CPC, about 64 vol. % of soybean oil, and about 19 vol. % of DiH₂O(designated herein as X8EC).

In alternative embodiments of the present invention, the formulationscomprise from about 1 to 2 vol. % of TRITON X-100, from about 1 to 2vol. % of TYLOXAPOL, from about 6 to 8 vol. % TBP, from about 0.5 to 1.0vol. % of CPC, from about 60 to 70 vol. % of oil (e.g., soybean), andabout 1 to 35 vol. % of aqueous phase (e.g., DiH₂O or PBS).Additionally, certain of these formulations may comprise from about 1 to5 vol. % of trypticase soy broth, from about 0.5 to 1.5 vol. % of yeastextract, about 5 mM L-alanine/Inosine, about 10 mM ammonium chloride,and from about 20-40 vol. % of liquid baby formula. In some of theembodiments comprising liquid baby formula, the formula comprises acasein hydrolysate (e.g., Neutramigen, or Progestimil, and the like). Insome of these embodiments, the inventive formulations further comprisefrom about 0.1 to 1.0 vol. % of sodium thiosulfate, and from about 0.1to 1.0 vol. % of sodium citrate. Other similar embodiments comprisingthese basic components employ phosphate buffered saline (PBS) as theaqueous phase. For example, one embodiment comprises about 2 vol. % ofTRITON X-100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol.% of CPC, about 64 vol. % of soybean oil, and about 23 vol. % of DiH₂O(designated herein as X2Y2EC). In still other embodiments, the inventiveformulation comprises about 2 vol. % of TRITON X-100, about 2 vol. %TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC, about 0.9 vol. %of sodium thiosulfate, about 0.1 vol. % of sodium citrate, about 64 vol.% of soybean oil, and about 22 vol. % of DiH₂O (designated herein asX2Y2PC STS1). In another similar embodiment, the formulations compriseabout 1.7 vol. % TRITON X-100, about 1.7 vol. % TYLOXAPOL, about 6.8vol. % TBP, about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 vol. %of soybean oil, and about 4.9 vol. % of DiH₂O (designated herein as 85%X2Y2PC/baby). In yet another embodiment of the present invention, theformulations comprise about 1.8 vol. % of TRITON X-100, about 1.8 vol. %of TYLOXAPOL, about 7.2 vol. % of TBP, about 0.9 vol. % of CPC, about 5mM L-alanine/Inosine, about 10 mM ammonium chloride, about 57.6 vol. %of soybean oil, and the remainder vol. % of 0.1×PBS (designated hereinas 90% X2Y2 PC/GE). In still another embodiment, the formulationscomprise about 1.8 vol. % of TRITON X-100, about 1.8 vol. % ofTYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol. % of CPC, and about 3vol. % trypticase soy broth, about 57.6 vol. % of soybean oil, and about27.7 vol. % of DiH₂O (designated herein as 90% X2Y2PC/TSB). In anotherembodiment of the present invention, the formulations comprise about 1.8vol. % TRITON X-100, about 1.8 vol. % TYLOXAPOL, about 7.2 vol. % TBP,about 0.9 vol. % CPC, about 1 vol. % yeast extract, about 57.6 vol. % ofsoybean oil, and about 29.7 vol. % of DiH₂O (designated herein as 90%X2Y2PC/YE).

In some embodiments of the present invention, the inventive formulationscomprise about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean or oliveoil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS).In a particular embodiment of the present invention, the inventiveformulations comprise about 3 vol. % of TYLOXAPOL, about 8 vol. % ofTBP, and about 1 vol. % of CPC, about 64 vol. % of soybean, and about 24vol. % of DiH₂O (designated herein as Y3PC).

In some embodiments of the present invention, the inventive formulationscomprise from about 4 to 8 vol. % of TRITON X-100, from about 5 to 8vol. % of TBP, about 30 to 70 vol. % of oil (e.g., soybean or oliveoil), and about 0 to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS).Additionally, certain of these embodiments further comprise about 1 vol.% of CPC, about 1 vol. % of benzalkonium chloride, about 1 vol. %cetylyridinium bromide, about 1 vol. % cetyldimethyletylammoniumbromide, 500 μM EDTA, about 10 mM ammonium chloride, about 5 mM Inosine,and about 5 mM L-alanine. For example, in certain of these embodiments,the inventive formulations comprise about 8 vol. % of TRITON X-100,about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 20 vol.% of DiH₂O (designated herein as X8P). In another embodiment of thepresent invention, the inventive formulations comprise about 8 vol. % ofTRITON X-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol. % ofsoybean oil, and about 19 vol. % of DiH₂O (designated herein as X8PC).In still another embodiment, the formulations comprise about 8 vol. %TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 50vol. % of soybean oil, and about 33 vol. % of DiH₂O (designated hereinas ATB-X1001). In yet another embodiment, the formulations compriseabout 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % ofCPC, about 50 vol. % of soybean oil, and about 32 vol. % of DiH₂O(designated herein as ATB-X002). Another embodiment of the presentinvention comprises about 4 vol. % TRITON X-100, about 4 vol. % of TBP,about 0.5 vol. % of CPC, about 32 vol. % of soybean oil, and about 59.5vol. % of DiH₂O (designated herein as 50% X8PC). Still another relatedembodiment comprises about 8 vol. % of TRITON X-100, about 8 vol. % ofTBP, about 0.5 vol. % CPC, about 64 vol. % of soybean oil, and about19.5 vol. % of DiH₂O (designated herein as X8PC_(1/2)). In someembodiments of the present invention, the inventive formulationscomprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2vol. % of CPC, about 64 vol. % of soybean oil, and about 18 vol. % ofDiH₂O (designated herein as X8PC2). In other embodiments, the inventiveformulations comprise about 8 vol. % of TRITON X-100, about 8% of TBP,about 1% of benzalkonium chloride, about 50 vol. % of soybean oil, andabout 33 vol. % of DiH₂O (designated herein as X8P BC). In analternative embodiment of the present invention, the formulationcomprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1vol. % of cetylyridinium bromide, about 50 vol. % of soybean oil, andabout 33 vol. % of DiH₂O (designated herein as X8P CPB). In anotherexemplary embodiment of the present invention, the formulations compriseabout 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % ofcetyldimethyletylammonium bromide, about 50 vol. % of soybean oil, andabout 33 vol. % of DiH₂O (designated herein as X8P CTAB). In stillfurther embodiments, the present invention comprises about 8 vol. % ofTRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 500 μMEDTA, about 64 vol. % of soybean oil, and about 15.8 vol. % DiH₂O(designated herein as X8PC EDTA). Additional similar embodimentscomprise 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. %of CPC, about 10 mM ammonium chloride, about 5 mM Inosine, about 5 mML-alanine, about 64 vol. % of soybean oil, and about 19 vol. % of DiH₂Oor PBS (designated herein as X8PC GE_(1x)). In another embodiment of thepresent invention, the inventive formulations further comprise about 5vol. % of TRITON X-100, about 5% of TBP, about 1 vol. % of CPC, about 40vol. % of soybean oil, and about 49 vol. % of DiH₂O (designated hereinas X5P₅C).

In some embodiments of the present invention, the inventive formulationscomprise about 2 vol. % TRITON X-100, about 6 vol. % TYLOXAPOL, about 8vol. % ethanol, about 64 vol. % of soybean oil, and about 20 vol. % ofDiH₂O (designated herein as X2Y6E).

In an additional embodiment of the present invention, the formulationscomprise about 8 vol. % of TRITON X-100, and about 8 vol. % of glycerol,about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15to 25 vol. % of aqueous phase (e.g., DiH₂O or PBS). Certain relatedembodiments further comprise about 1 vol. % L-ascorbic acid. Forexample, one particular embodiment comprises about 8 vol. % of TRITONX-100, about 8 vol. % of glycerol, about 64 vol. % of soybean oil, andabout 20 vol. % of DiH₂O (designated herein as X8G). In still anotherembodiment, the inventive formulations comprise about 8 vol. % of TRITONX-100, about 8 vol. % of glycerol, about 1 vol. % of L-ascorbic acid,about 64 vol. % of soybean oil, and about 19 vol. % of DiH₂O (designatedherein as X8GV_(c)).

In still further embodiments, the inventive formulations comprise about8 vol. % of TRITON X-100, from about 0.5 to 0.8 vol. % of TWEEN 60, fromabout 0.5 to 2.0 vol. % of CPC, about 8 vol. % of TBP, about 60 to 70vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol. % ofaqueous phase (e.g., DiH₂O or PBS). For example, in one particularembodiment the formulations comprise about 8 vol. % of TRITON X-100,about 0.70 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % ofTBP, about 64 vol. % of soybean oil, and about 18.3 vol. % of DiH₂O(designated herein as X8W60PC_(L)). Another related embodiment comprisesabout 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 1vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil,and about 18.29 vol. % of DiH₂O (designated herein as W60_(0.7)X8PC). Inyet other embodiments, the inventive formulations comprise from about 8vol. % of TRITON X-100, about 0.7 vol. % of TWEEN 60, about 0.5 vol. %of CPC, about 8 vol. % of TBP, about 64 to 70 vol. % of soybean oil, andabout 18.8 vol. % of DiH₂O (designated herein as X8W60PC₂). In stillother embodiments, the present invention comprises about 8 vol. % ofTRITON X-100, about 0.71 vol. % of TWEEN 60, about 2 vol. % of CPC,about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 17.3vol. % of DiH₂O. In another embodiment of the present invention, theformulations comprise about 0.71 vol. % of TWEEN 60, about 1 vol. % ofCPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about25.29 vol. % of DiH₂O (designated herein as W60_(0.7)PC).

In another embodiment of the present invention, the inventiveformulations comprise about 2 vol. % of dioctyl sulfosuccinate, eitherabout 8 vol. % of glycerol, or about 8 vol. % TBP, in addition to, about60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 20 to 30vol. % of aqueous phase (e.g., DiH₂O or PBS). For example, oneembodiment of the present invention comprises about 2 vol. % of dioctylsulfosuccinate, about 8 vol. % of glycerol, about 64 vol. % of soybeanoil, and about 26 vol. % of DiH₂O (designated herein as D2G). In anotherrelated embodiment, the inventive formulations comprise about 2 vol. %of dioctyl sulfosuccinate, and about 8 vol. % of TBP, about 64 vol. % ofsoybean oil, and about 26 vol. % of DiH₂O (designated herein as D2P).

In still other embodiments of the present invention, the inventiveformulations comprise about 8 to 10 vol. % of glycerol, and about 1 to10 vol. % of CPC, about 50 to 70 vol. % of oil (e.g., soybean or oliveoil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS).Additionally, in certain of these embodiments, the compositions furthercomprise about 1 vol. % of L-ascorbic acid. For example, one particularembodiment comprises about 8 vol. % of glycerol, about 1 vol. % of CPC,about 64 vol. % of soybean oil, and about 27 vol. % of DiH₂O (designatedherein as GC). An additional related embodiment comprises about 10 vol.% of glycerol, about 10 vol. % of CPC, about 60 vol. % of soybean oil,and about 20 vol. % of DiH₂O (designated herein as GC10). In stillanother embodiment of the present invention, the inventive formulationscomprise about 10 vol. % of glycerol, about 1 vol. % of CPC, about 1vol. % of L-ascorbic acid, about 64 vol. % of soybean or oil, and about24 vol. % of DiH₂O (designated herein as GCV_(c)).

In some embodiments of the present invention, the inventive formulationscomprise about 8 to 10 vol. % of glycerol, about 8 to 10 vol. % of SDS,about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15to 30 vol. % of aqueous phase (e.g., DiH₂O or PBS). Additionally, incertain of these embodiments, the compositions further comprise about 1vol. % of lecithin, and about 1 vol. % of p-Hydroxybenzoic acid methylester. Exemplary embodiments of such formulations comprise about 8 vol.% SDS, 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about20 vol. % of DiH₂O (designated herein as S8G). A related formulationcomprises about 8 vol. % of glycerol, about 8 vol. % of SDS, about 1vol. % of lecithin, about 1 vol. % of p-Hydroxybenzoic acid methylester, about 64 vol. % of soybean oil, and about 18 vol. % of DiH₂O(designated herein as S8GL1B1).

In yet another embodiment of the present invention, the inventiveformulations comprise about 4 vol. % of TWEEN 80, about 4 vol. % ofTYLOXAPOL, about 1 vol. % of CPC, about 8 vol. % of ethanol, about 64vol. % of soybean oil, and about 19 vol. % of DiH₂O (designated hereinas W₈₀4Y4EC).

In some embodiments of the present invention, the inventive formulationscomprise about 0.01 vol. % of CPC, about 0.08 vol. % of TYLOXAPOL, about10 vol. % of ethanol, about 70 vol. % of soybean oil, and about 19.91vol. % of DiH₂O (designated herein as Y.08EC.01).

In yet another embodiment of the present invention, the inventiveformulations comprise about 8 vol. % of sodium lauryl sulfate, and about8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol.% of DiH₂O (designated herein as SLS8G).

C. Additional Formulations

The specific formulations described above are simply examples toillustrate the variety of compositions that find use in the presentinvention. The present invention contemplates that many variations ofthe above formulation, as well as additional nanoemulsions, find use inthe methods of the present invention. To determine if a candidateemulsion is suitable for use with the present invention, three criteriamay be analyzed. Using the methods and standards described herein,candidate emulsions can be easily tested to determine if they aresuitable. First, the desired ingredients are prepare using the methodsdescribed herein, to determine if an emulsion can be formed. If anemulsion cannot be formed, the candidate is rejected. For example, acandidate composition made of 4.5% sodium thiosulfate, 0.5% sodiumcitrate, 10% n-butanol, 64% soybean oil, and 21% DiH₂O did not form anemulsion.

Second, the candidate emulsion should form a stable emulsion. Anemulsion is stable if it remains in emulsion form for a sufficientperiod to allow its intended use. For example, for emulsions that are tobe stored, shipped, etc., it may be desired that the composition remainin emulsion form for months to years. Typical emulsions that arerelatively unstable, will lose their form within a day. For example, acandidate composition made of 8% 1-butanol, 5% Tween 10, 1% CPC, 64%soybean oil, and 22% DiH₂O did not form a stable emulsion. The followingcandidate emulsions were shown to be stable using the methods describedherein: 0.08% Triton X-100, 0.08% Glycerol, 0.01% CetylpyridiniumChloride, 99% Butter, and 0.83% diH₂O (designated herein as 1% X8GCButter); 0.8% Triton X-100, 0.8% Glycerol, 0.1% CetylpyridiniumChloride, 6.4% Soybean Oil, 1.9% diH₂O, and 90% Butter (designatedherein as 10% X8GC Butter); 2% W₂₀5EC, 1% Natrosol 250L NF, and 97%diH₂O (designated herein as 2% W₂₀5EC L GEL); 1% CetylpyridiniumChloride, 5% Tween 20, 8% Ethanol, 64% 70 Viscosity Mineral Oil, and 22%diH₂O (designated herein as W₂₀5EC 70 Mineral Oil); 1% CetylpyridiniumChloride, 5% Tween 20, 8% Ethanol, 64% 350 Viscosity Mineral Oil, and22% diH₂O (designated herein as W₂₀5EC 350 Mineral Oil). In someembodiments, nanoemulsions of the present invention are stable for overa week, over a month, or over a year.

Third, the candidate emulsion should have efficacy for its intended use.For example, an anti-bacterial emulsion should kill or disable bacteriato a detectable level or to a preferred kill level (e.g., 1 log, 2 log,3 log, 4 log, . . . reduction). As shown herein, certain emulsions ofthe present invention have efficacy against specific microorganisms, butnot against others. Using the methods described herein, one is capableof determining the suitability of a particular candidate emulsionagainst the desired microorganism. Generally, this involves exposing themicroorganism to the emulsion for one or more time periods in aside-by-side experiment with the appropriate control samples (e.g., anegative control such as water) and determining if, and to what degree,the emulsion kills or disable the microorganism. For example, acandidate composition made of 1% ammonium chloride, 5% Tween 20, 8%ethanol, 64% soybean oil, and 22% DiH₂O was shown not to be an effectiveemulsion. The following candidate emulsions were shown to be effectiveusing the methods described herein: 5% Tween 20, 5% CetylpyridiniumChloride, 10% Glycerol, 60% Soybean Oil, and 20% diH₂O (designatedherein as W₂₀5GC5); 1% Cetylpyridinium Chloride, 5% Tween 20, 10%Glycerol, 64% Soybean Oil, and 20% diH₂O (designated herein as W₂₀5GC);1% Cetylpyridinium Chloride, 5% Tween 20, 8% Ethanol, 64% Olive Oil, and22% diH₂O (designated herein as W₂₀5EC Olive Oil); 1% CetylpyridiniumChloride, 5% Tween 20, 8% Ethanol, 64% Flaxseed Oil, and 22% diH₂O(designated herein as W₂₀5EC Flaxseed Oil); 1% Cetylpyridinium Chloride,5% Tween 20, 8% Ethanol, 64% Corn Oil, and 22% diH₂O (designated hereinas W₂₀5EC Corn Oil); 1% Cetylpyridinium Chloride, 5% Tween 20, 8%Ethanol, 64% Coconut Oil, and 22% diH₂O (designated herein as W₂₀5ECCoconut Oil); 1% Cetylpyridinium Chloride, 5% Tween 20, 8% Ethanol, 64%Cottonseed Oil, and 22% diH₂O (designated herein as W₂₀5EC CottonseedOil); 8% Dextrose, 5% Tween 10, 1% Cetylpyridinium Chloride, 64% SoybeanOil, and 22% diH₂O (designated herein as W₂₀5C Dextrose); 8% PEG 200, 5%Tween 10, 1% Cetylpyridinium Chloride, 64% Soybean Oil, and 22% diH₂O(designated herein as W₂₀5C PEG 200); 8% Methanol, 5% Tween 10, 1%Cetylpyridinium Chloride, 64% Soybean Oil, and 22% diH₂O (designatedherein as W₂₀5C Methanol); 8% PEG 1000, 5% Tween 10, 1% CetylpyridiniumChloride, 64% Soybean Oil, and 22% diH₂O (designated herein as W₂₀5C PEG1000); 2% W₂₀5EC, 2% Natrosol 250H NF, and 96% diH₂O (designated hereinas 2% W₂₀5EC Natrosol 2, also called 2% W₂₀5EC GEL); 2% W₂₀5EC, 1%Natrosol 250H NF, and 97% diH₂O (designated herein as 2% W₂₀5EC Natrosol1); 2% W₂₀5EC, 3% Natrosol 250H NF, and 95% diH₂O (designated herein as2% W₂₀5EC Natrosol 3); 2% W₂₀5EC, 0.5% Natrosol 250H NF, and 97.5% diH₂O(designated herein as 2% W₂₀5EC Natrosol 0.5); 2% W₂₀5EC, 2% Methocel A,and 96% diH₂O (designated herein as 2% W₂₀5EC Methocel A); 2% W₂₀5EC, 2%Methocel K, and 96% diH₂O (designated herein as 2% W₂₀5EC Methocel K);2% Natrosol, 0.1% X8PC, 0.1×PBS, 5 mM L-alanine, 5 mM Inosine, 10 mMAmmonium Chloride, and diH₂O (designated herein as 0.1% X8PC/GE+2%Natrosol); 2% Natrosol, 0.8% Triton X-100, 0.8% Tributyl Phosphate, 6.4%Soybean Oil, 0.1% Cetylpyridinium Chloride, 0.1×PBS, 5 mM L-alanine, 5mM Inosine, 10 mM Ammonium Chloride, and diH₂O (designated herein as 10%X8PC/GE+2% Natrosol); 1% Cetylpyridinium Chloride, 5% Tween 20, 8%Ethanol, 64% Lard, and 22% diH₂O (designated herein as W₂₀5EC Lard); 1%Cetylpyridinium Chloride, 5% Tween 20, 8% Ethanol, 64% Mineral Oil, and22% diH₂O (designated herein as W₂₀5EC Mineral Oil); 0.1%Cetylpyridinium Chloride, 2% Nerolidol, 5% Tween 20, 10% Ethanol, 64%Soybean Oil, and 18.9% diH₂O (designated herein as W₂₀5EC_(0.1)N); 0.1%Cetylpyridinium Chloride, 2% Farnesol, 5% Tween 20, 10% Ethanol, 64%Soybean Oil, and 18.9% diH₂O (designated herein as W₂₀5EC_(0.1)F); 0.1%Cetylpyridinium Chloride, 5% Tween 20, 10% Ethanol, 64% Soybean Oil, and20.9% diH₂O (designated herein as W₂₀5EC_(0.1)); 10% CetylpyridiniumChloride, 8% Tributyl Phosphate, 8% Triton X-100, 54% Soybean Oil, and20% diH₂O (designated herein as X8PC₁₀); 5% Cetylpyridinium Chloride, 8%Triton X-100, 8% Tributyl Phosphate, 59% Soybean Oil, and 20% diH₂O(designated herein as X8PC₅); 0.02% Cetylpyridinium Chloride, 0.1% Tween20, 10% Ethanol, 70% Soybean Oil, and 19.88% diH₂O (designated herein asW₂₀0.1EC_(0.02)); 1% Cetylpyridinium Chloride, 5% Tween 20, 8% Glycerol,64% Mobil 1, and 22% diH₂O (designated herein as W₂₀5GC Mobil 1); 7.2%Triton X-100, 7.2% Tributyl Phosphate, 0.9% Cetylpyridinium Chloride,57.6% Soybean Oil, 0.1×PBS, 5 mM L-alanine, 5 mM Inosine, 10 mM AmmoniumChloride, and 25.87% diH₂O (designated herein as 90% X8PC/GE); 7.2%Triton X-100, 7.2% Tributyl Phosphate, 0.9% Cetylpyridinium Chloride,57.6% Soybean Oil, 1% EDTA, 5 mM L-alanine, 5 mM Inosine, 10 mM AmmoniumChloride, 0.1×PBS, and diH₂O (designated herein as 90% X8PC/GE EDTA);and 7.2% Triton X-100, 7.2% Tributyl Phosphate, 0.9% CetylpyridiniumChloride, 57.6% Soybean Oil, 1% Sodium Thiosulfate, 5 mM L-alanine, 5 mMInosine, 10 mM Ammonium Chloride, 0.1×PBS, and diH₂O (designated hereinas 90% X8PC/GE STS).

D. Non-Toxic, Non-Irritant, Non-Corrosive Formulations

In preferred embodiments of the present invention, the nanoemulsions arenon-toxic (e.g., to humans, plants, or animals), non-irritant (e.g., tohumans, plants, or animals), and non-corrosive (e.g., to humans, plants,or animals or the environment), while possessing potency against a broadrange of microorganisms including bacteria, fungi, viruses, and spores.While a number of the above described nanoemulsions meet thesequalifications, the following description provides a number of preferrednon-toxic, non-irritant, non-corrosive, anti-microbial nanoemulsions ofthe present invention (hereinafter in this section referred to as“non-toxic nanoemulsions”).

In some embodiments the non-toxic nanoemulsions comprise surfactantlipid preparations (SLPs) for use as broad-spectrum antimicrobial agentsthat are effective against bacteria and their spores, enveloped viruses,and fungi. In preferred embodiments, these SLPs comprises a mixture ofoils, detergents, solvents, and cationic halogen-containing compounds inaddition to several ions that enhance their biocidal activities. TheseSLPs are characterized as stable, non-irritant, and non-toxic compoundscompared to commercially available bactericidal and sporicidal agents,which are highly irritant and/or toxic.

Ingredients for use in the non-toxic nanoemulsions include, but are notlimited to: detergents (e.g., TRITON X-100 [5-15%] or other members ofthe TRITON family, TWEEN 60 [0.5-2%] or other members of the TWEENfamily, or TYLOXAPOL [1-10%]); solvents (e.g., tributyl phosphate[5-15%]); alcohols (e.g., ethanol [5-15%] or glycerol [5-15%]); oils(e.g., soybean oil [40-70%]); cationic halogen-containing compounds(e.g., cetylpyridinium chloride [0.5-2%], cetylpyridinium bromide[0.5-2%]), or cetyldimethylethyl ammonium bromide [0.5-2%]); quaternaryammonium compounds (e.g., benzalkonium chloride [0.5-2%],N-alkyldimethylbenzyl ammonium chloride [0.5-2%]); ions (calciumchloride [1 mM-40 mM], ammonium chloride [1 mM-20 mM], sodium chloride[5 mM-200 mM], sodium phosphate [1 mM-20 mM]); nucleosides (e.g.,inosine [501M-20 mM]); and amino acids (e.g., L-alanine [50 μM-20 mM]).Emulsions are prepared, for example, by mixing in a high shear mixer for3-10 minutes. The emulsions may or may not be heated before mixing at82° C. for 1 hour.

Quaternary ammonium compounds for use in the present include, but arenot limited to, N-alkyldimethyl benzyl ammonium saccharinate;1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol; 1-Decanaminium,N-decyl-N,N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride;2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammoniumchloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzylammonium chloride; alkyl 1 or 3 benzyl-1-(2-hydroxethyl)-2-imidazoliniumchloride; alkyl bis(2-hydroxyethyl) benzyl ammonium chloride; alkyldemethyl benzyl ammonium chloride; alkyl dimethyl 3,4-dichlorobenzylammonium chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammoniumchloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzylammonium chloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzylammonium chloride; alkyl dimethyl benzyl ammonium chloride (100% C14);alkyl dimethyl benzyl ammonium chloride (100% C16); alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12); alkyl dimethyl benzylammonium chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammoniumchloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride(58% C14, 28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12); alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14);alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyldimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl dimethylbenzyl ammonium chloride (67% C12, 24% C14); alkyl dimethyl benzylammonium chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammoniumchloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93%C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18);alkyl dimethyl benzyl ammonium chloride (and) didecyl dimethyl ammoniumchloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids);alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzylammonium chloride (C12-C18); alkyl dimethyl benzyl and dialkyl dimethylammonium chloride; alkyl dimethyl dimethybenzyl ammonium chloride; alkyldimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyldimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as inthe fatty acids of soybean oil); alkyl dimethyl ethylbenzyl ammoniumchloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyldimethyl isoproylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3%C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1%C12); alkyl trimethyl ammonium chloride (90% C18, 10% C16);alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); Di-(C8-10)-alkyldimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyldimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkylmethyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride;diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammoniumchloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride;dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyldimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazoliniumchloride; hexahydro-1,3,5-thris(2-hydroxyethyl)-s-triazine;myristalkonium chloride (and) Quat RNIUM 14;N,N-Dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethylbenzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammoniumchloride; n-tetradecyl dimethyl benzyl ammonium chloride monohydrate;octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammoniumchloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride;oxydiethylenebis (alkyl dimethyl ammonium chloride); quaternary ammoniumcompounds, dicoco alkyldimethyl, chloride; trimethoxysily propyldimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammoniumchloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyldimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethylbenzylammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

In general, the preferred non-toxic nanoemulsions are characterized bythe following: they are approximately 200-800 nm in diameter, althoughboth larger and smaller diameter nanoemulsions are contemplated; thecharge depends on the ingredients; they are stable for relatively longperiods of time (e.g., up to two years), with preservation of theirbiocidal activity; they are non-irritant and non-toxic compared to theirindividual components due, at least in part, to their oil contents thatmarkedly reduce the toxicity of the detergents and the solvents; theyare effective at concentrations as low as 0.1%; they have antimicrobialactivity against most vegetative bacteria (including Gram-positive andGram-negative organisms), fungi, and enveloped and nonenveloped virusesin 15 minutes (e.g., 99.99% killing); and they have sporicidal activityin 1-4 hours (e.g., 99.99% killing) when produced with germinationenhancers.

III. Properties and Activities

The specific compositions of the present invention possess a range ofbeneficial activities and properties. A number of the exemplarybeneficial properties and activities are set forth below: A)Microbicidal and Microbistatic Activity; B) Sporicidial and SporistaticActivity: C) Viricidal and Viralstatic Activity; D) Fungicidal andFungistatic Activity; and E) In vivo Effects. Additionally, FIG. 31A-Cprovides properties of certain exemplary formulations of the presentinvention. In some preferred embodiments, the nanoemulsions of thepresent invention have broad spectrum killing activities, whereby theykill or disable, two or more of: 1) bacteria (gram positive and gramnegative), 2) viruses, 3) fungi, and 4) spores (e.g., 1 log, 2 log, 3log, 4 log, . . . reduction).

A. Microbicidal and Microbistatic Activity

The methods of the present invention can be used to rapidly inactivatebacteria. In certain embodiments, the compositions are particularlyeffective at inactivating Grain positive bacteria. In preferredembodiments, the inactivation of bacteria occurs after about five to tenminutes. Thus, bacteria may be contacted with an emulsion according tothe present invention and will be inactivated in a rapid and efficientmanner. It is expected that the period of time between the contactingand inactivation may be as little as 5-10 minutes or less where thebacteria is directly exposed to the emulsion. However, it is understoodthat when the emulsions of the present invention are employed in atherapeutic context and applied systemically, the inactivation may occurover a longer period of time including, but not limited to, 5, 10, 15,20, 25, 30, 60 minutes post application. Further, in additionalembodiments it may be that the inactivation may take two, three, four,five or six hours to occur.

In other embodiments, the compositions and methods of the invention canalso rapidly inactivate certain Gram negative bacteria. In someembodiments, the bacteria inactivating emulsions are premixed with acompound that increases the interaction of the emulsion by the cellwall. The use of these enhancers in the compositions of the presentinvention is discussed herein below. It should be noted that certainemulsions especially those comprising enhancers are effective againstcertain Gram positive and negative bacteria and may be administeredorally where they will come in contact with necessary gut bacteria.

In specific embodiments, the present invention has shown that theemulsions of the present invention have potent, selective biocidalactivity with minimal toxicity against vegetative bacteria. BCTP washighly effective against B. cereus, B. circulans and B. megaterium, C.perfringens, H. influenzae, N. gonorrhoeae, S. agalactiae, S. pneumonia,S. pyogenes and V. cholerae classical and Eltor (FIG. 26). Thisinactivation starts immediately on contact and is complete within 15 to30 minutes for most of the susceptible microorganisms.

FIG. 31A shows the effectiveness of a number of exemplary nanoemulsionsof the present invention against E. coli.

B. Sporicidial and Sporistatic Activity

In certain specific embodiments, the present invention has demonstratedthat the emulsions of the present invention have sporicidal activity.Without being bound to any theory (an understanding of the mechanism isnot necessary to practice the present invention, and the presentinvention is not limited to any particular mechanism), it is proposedthe that the sporicidal ability of these emulsions occurs throughinitiation of germination without complete reversion to the vegetativeform leaving the spore susceptible to disruption by the emulsions. Theinitiation of germination could be mediated by the action of theemulsion or its components.

The results of electron microscopy studies show disruption of the sporecoat and cortex with disintegration of the core contents following BCTPtreatment. Sporicidal activity appears to be mediated by both the TRITONX-100 and tri-n-butyl phosphate components since nanoemulsions lackingeither component are inactive in vivo. This unique action of theemulsions, which is similar in efficiency to 1% breach, is interestingbecause Bacillus spores are generally resistant to most disinfectantsincluding many commonly used detergents (Russell, Clin. Micro. 3; 99[1990]).

The present invention demonstrates that mixing BCTP with B. cereusspores before injecting into mice prevented the pathological effect ofB. cereus. Further, the present invention shows that BCTP treatment ofsimulated wounds contaminated with B. cereus spores markedly reduced therisk of infection and mortality in mice. The control animals, that wereinjected with BCTP alone diluted 1:10, did not show any inflammatoryeffects proving that BCTP does not have cutaneous toxicity in mice.These results suggest that immediate treatment of spores prior to orfollowing exposure can effectively reduce the severity of tissue damageof the experimental cutaneous infection.

Other experiments conducted during the development of the presentinvention compared the effects of BCTP and other emulsions derived fromBCTP to inactivate different Bacillus spores. BCTP diluted up to 1:1000(v/v) inactivated more than 90% of B. anthracis spores in four hours,and was also sporicidal against three other Bacillus species through theapparent disruption of spore coat. X₈W₆₀PC diluted 1:1000 had moresporicidal activity against B. anthracis, B. cereus, and B. subtilis andhad an onset of action in less than 30 minutes. In mice, mixing BCTPwith B. cereus before subcutaneous injection or wound irrigation withBCTP 1 hour following spore inoculation resulted in over 98% reductionin skin lesion size. Mortality was reduced 4-fold in the latterexperiment. The present compositions are stable, easily dispersed,non-irritant and nontoxic compared to the other available sporicidalagents.

The bacteria-inactivating oil-in-water emulsions used in the methods ofthe present invention can be used to inactivate a variety of bacteriaand bacterial spores upon contact. For example, the presently disclosedemulsions can be used to inactivate Bacillus including B. cereus, B.circulans and B. megatetium, also including Clostridium (e.g., C.botulinum and C. tetani). The methods of the present invention may beparticularly useful in inactivating certain biological warfare agents(e.g., B. anthracis). In addition, the formulations of the presentinvention also find use in combating C. perftingens, H. influenzae, N.gonorrhoeae, S. agalactiae, S. pneumonia, S. pyogenes and V. choleraeclassical and Eltor (FIG. 26).

BCTP contains TRITON X-100 while SS and W₈₀8P contain TWEEN 60, and NNcontained nonoxynol-9 surfactant. Each is a non-ionic surfactant, butdiffers in its chemistry and biological characteristics. Nonoxynol-9 hasstrong spermicidal activity and it is widely used as a component ofvaginally delivered contraceptive products (Lee, 1996). It has beenclaimed to have virucidal effect against enveloped viruses (Hermonat etal., 1992; Zeitlin et al., 1997). However, nanoxynol-9 has not beenshown to be effective against nonenveloped viruses (Hermonat et al.,1992).

FIG. 31B shows the effectiveness of a number of exemplary nanoemulsionsof the present invention against B. globigii spores.

C. Viricidal and Viralstatic Activity

In additional embodiments, it was demonstrated that the nanoemulsioncompositions of the present invention have anti-viral properties. Theeffect of these emulsions on viral agents was monitored using plaquereduction assay (PRA), cellular enzyme-linked immunosorbent assay(ELISA), P-galactosidase assay, and electron microscopy (EM) and thecellular toxicity of lipid preparations was assessed using a(4,5-dimethylthiazole-2-yl)-2,5 diphenyltetrazolium (MTT) staining assay(Mosmann 1983).

There was a marked reduction of influenza A infectivity of MDCK cells asmeasured by cellular ELISA with subsequent confirmation by PRA. BCTP andSS at dilution 1:10 reduced virus infectivity over 95%. Two otheremulsions showed only intermediate effects on the virus reducinginfectivity by approximately 40% at dilution 1:10. BCTP was the mostpotent preparation and showed undiminished virucidal effect even atdilution 1:100. Kinetic studies showed that 5 min incubation of viruswith BCTP at 1:10 dilution completely abolished its infectivity. TRITONX-100, an active compound of BCTP, at dilution 1:5000 only partiallyinhibited the infectivity of virus as compared to BCTP, indicating thatthe nanoemulsion itself contributes to the anti-viral efficacy. Tofurther examine the anti-viral properties of BCTP, its action onnon-enveloped viruses was investigated. The BCTP treatment did notaffect the replication of lacZ adenovirus construct in 293 cells asmeasured using β-galactosidase assay. When examined with EM, influenza Avirus was completely disrupted after incubation with BCTP whileadenovirus remained intact.

In addition, pre-incubation of virus with 10% and 1% BCTP in PBScompletely eliminates herpes, sendai, sindbis and vaccinia viruses asassessed by plaque reduction assays (FIG. 27). Time course analysesshowed the onset of inactivation to be rapid and complete within 5minutes of incubation with 10% BCTP and within 30 minutes with 1% BCTP.Adenovirus treated with different dilutions of BCTP showed no reductionin infectivity.

The efficacy of certain BCTP based compositions against various viralonslaught and their minimal toxicity to mucous membranes demonstratetheir potential as effective disinfectants and agents for prevention ofdiseases resulting from infection with enveloped viruses.

In some embodiments, the nanoemulsions of the present invention are usedin conjunction with a low pH buffer. Such nanoemulsions find use asrapid killers of viruses (e.g., rhinovirus or other picornaviruses).

FIG. 31C shows the effectiveness of a number of exemplary nanoemulsionsof the present invention against influenza A.

D. Fungicidal and Fungistatic Activity

Yet another property of the nanoemulsions of the present invention isthat they possess antifungal activity. Common agents of fungalinfections include various species of the genii Candida and Aspergillus,and types thereof, as well as others. While external fungus infectionscan be relatively minor, systemic fungal infections can give rise toserious medical consequences. There is an increasing incidence of fungalinfections in humans, attributable in part to an increasing number ofpatients having impaired immune systems. Fungal disease, particularlywhen systemic, can be life threatening to patients having an impairedimmune system.

Experiments conducted during the development of the present inventionhave shown that 1% BCTP has a greater than 92% fungistatic activity whenapplied to Candida albicans. Candida was grown at 37° C. overnight.Cells were then washed and counted using a hemacytometer. A known amountof cells were mixed with different concentrations of BCTP and incubatedfor 24 hours. The Candida was then grown on dextrose agar, incubatedovernight, and the colonies were counted. The fungistatic effect of theBCTP was determined as follows:

${{Fungistatic}\mspace{14mu} {effect}\mspace{11mu} ({FSE})} = {1 - {\frac{{\# \mspace{14mu} {of}\mspace{14mu} {treated}\mspace{14mu} {cells}} - {{Initial}\mspace{14mu} \# \mspace{14mu} {of}\mspace{14mu} {cells}}}{{\# \mspace{14mu} {of}\mspace{14mu} {untreated}\mspace{14mu} {cells}} - {{Initial}\mspace{14mu} \# \mspace{14mu} {of}\mspace{14mu} {cells}}} \times 100}}$

One of skill in the art will be able to take the formulations of thepresent invention and place them into appropriate formulations for thetreatment of fungal disease. The nanoemulsions of the present inventionfind use in combatting infections such as athletes foot, candidosis andother acute or systemic fungal infections.

E. In Vivo Effects

Animal studies demonstrated the protective and therapeutic effect of thepresent compositions and methods. Bacillus cereus infection inexperimental animals has been used previously as a model system for thestudy of anthrax (See e.g., Burdon and Wende, J. Infect. Diseas.170(2):272 [1960]; Lamanna and Jones, J. Bact. 85:532 [1963]; and Burdonet al., J. Infect. Diseas. 117:307 [1967]). The disease syndrome inducedin animals experimentally infected with B. cereus is similar to anthrax(Drobniewski, Clin. microbio. Rev. 6:324 [1993]; and Fritz et al., Lab.Invest. 73:691 [1995]). Experiments conducted during the development ofthe present invention demonstrated that mixing BCTP with B. cereusspores before injecting into mice prevented the pathological effect ofB. cereus. Further, it was demonstrated that BCTP treatment of simulatedwounds contaminated with B. cereus spores markedly reduced the risk ofinfection and mortality in mice. The control animals, which wereinjected with BCTP alone diluted 1:10, did not show any inflammatoryeffects proving that BCTP does not have cutaneous toxicity in mice.These results suggest that immediate treatment of spores prior to orfollowing exposure can effectively reduce the severity of tissue damageof the experimental cutaneous infection.

In a particular example, Guinea Pigs were employed as experimentalanimals for the study of C. perftingens infection. A 1.5 cm skin woundwas made, the underlying muscle was crushed and infected with 5×10⁷ cfuof C perftingens without any further treatment. Another group wasinfected with the same number of bacteria, then 1 hour later it wasirrigated with either saline or BCTP to simulate post-exposuredecontamination. Irrigation of experimentally infected wounds withsaline did not result in any apparent benefit. However, BCTP irrigationof the wound infected with C. perfingens showed marked reduction ofedema, inflammatory reaction and necrosis. As such, it was demonstratedthat certain formulations of the present invention can be used to combata bacterial infection.

Further, a subcutaneous injection of 10% BCTP did not cause distress inexperimental animals and resulted in no gross histological tissuedamage. All rats in the oral toxicity study showed weight gain over thestudy period. No adverse clinical signs were noted and all tissuesappeared within normal limits on gross examination. Bacterial culturesfrom the stools of treated animals were not significantly different fromthose of untreated animals.

IV. Exemplary Uses

Set forth below are a number of exemplary uses for the compositionsdisclosed herein: A) Pharmaceuticals and Therapeutics; B)Decontamination and Sterilization; C) Food Preparation; and D) Kits, aswell as a description of methods and systems for the E) Modification,Preparation, and Delivery of the compositions of the present invention.

A. Pharmaceuticals and Therapeutics

The present invention contemplates formulations that may be employed inpharmaceutical and therapeutic compositions and applications suitablefor combatting and/or treating microbial infections. Such compositionsmay be employed to reduce infection, kill microbes, inhibit microbialgrowth or otherwise abrogate the deleterious effects of microbialinfection.

For in vivo applications, the compositions can be administered in anyeffective pharmaceutically acceptable form to warm blooded animals,including human and animal subjects. Generally, this entails preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

Particular examples of pharmaceutically acceptable forms include but arenot limited to oral, nasal, buccal, rectal, vaginal, topical or nasalspray or in any other form effective to deliver active compositions ofthe present invention to a site of microorganism infection. In preferredembodiments, the route of administration is designed to obtain directcontact of the compositions with the infecting microorganisms. In otherembodiments, administration may be by orthotopic, intradermal,subcutaneous, intramuscular or intraperitoneal injection. Thecompositions may also be administered to subjects parenterally orintraperitonealy. Such compositions would normally be administered aspharmaceutically acceptable compositions. Except insofar as anyconventional pharmaceutically acceptable media or agent is incompatiblewith the emulsions of the present invention, the use of knownpharmaceutically acceptable media and agents in these particularembodiments is contemplated. In additional embodiments, supplementaryactive ingredients also can be incorporated into the compositions.

For topical applications, the pharmaceutically acceptable carrier maytake the form of a liquid, cream, foam, lotion, or gel, and mayadditionally comprise organic solvents, emulsifiers, gelling agents,moisturizers, stabilizers, surfactants, wetting agents, preservatives,time release agents, and minor amounts of humectants, sequesteringagents, dyes, perfumes, and other components commonly employed inpharmaceutical compositions for topical administration.

Tablet and dosage forms of the compositions in which the emulsions areformulated for oral or topical administration include liquid capsules,and suppositories. In solid dosage forms for oral administration, thecompositions may be admixed with one or more substantially inert diluent(e.g., sucrose, lactose, or starch, and the like) and may additionallycomprise lubricating agents, buffering agents, enteric coatings, andother components well known to those skilled in the art.

In another embodiment of the invention, the compositions of theinvention may be specifically designed for in vitro applications, suchas disinfecting or sterilization of medical instruments and devices,contact lenses and the like, particularly when the devices or lenses areintended to be used in contact with a patient or wearer. For example,the compositions may be used to cleanse and decontaminate medical andsurgical instruments and supplies prior to contacting a subject.Additionally, the compositions may be used to post-operatively, or afterany invasive procedure, to help minimize the occurrence of postoperative infections. In especially preferred embodiments, thecompositions are administered to subjects with compromised orineffective immunological defenses (e.g., the elderly and the veryyoung, burn and trauma victims, and those infected with HIV and thelike). For applications of this type, the compositions may beconveniently provided in the form of a liquid, foam, paste or gel andmay be provided with emulsifiers, surfactants, buffering agents, wettingagents, preservatives, metal ions, antibiotics and other componentscommonly found in compositions of this type.

In some embodiments, the compositions are used in association with organor artificial tissue transplantation or maintenance. For example, thecomposition may be used on the surface of a transplanted organ tosterilize the organ. The compositions may also be used in conductionwith organ preservation or storage solutions (e.g., VIASPAN, BarrLaboratories).

In other embodiments, the compositions may be impregnated intoabsorptive materials, such as sutures, bandages, and gauze, or coatedonto the surface of solid phase materials, such as surgical staples,zippers and catheters to deliver the compositions to a site for theprevention of microbial infection. Other delivery systems of this typewill be readily apparent to those skilled in the art.

In yet another embodiment, the compositions can be used in the personalhealth care industry in deodorants, soaps, acne/dermatophyte treatmentagents, treatments for halitosis, treatments for vaginal yeastinfections, and the like. The compositions can also be used to treatother internal and external microbial infections (e.g., influenza, H.simplex, toe-nail fungus, etc.). In these applications, the emulsionscan be formulated with therapeutic carriers as described above. In someembodiments, the nanoemulsions of the present invention are formulatedinto gels, wherein the gels are applied topically.

In certain embodiments, the antimicrobial compositions and methods ofthe present invention also include a variety of combination therapies.For example, often single antimicrobial agents are much less effectiveat inhibiting microbes than are several agents employed in conjunctionwith each other. This approach is often advantageous in avoiding theproblems encountered as a result of multidrug resistance. This isparticularly prevalent in bacteria that have drug transporters thatmediate the efflux of drugs from the organism. The present inventionfurther contemplates the use of the present methods and compositions insuch combination therapies.

In some embodiments, the nanoemulsion compositions of the presentinvention are used as a delivery system for another agent (e.g., apharmaceutical agent). In some embodiments, the agent has antimicrobialproperties. In such embodiments, the nanoemulsions of the presentinvention increase the antimicrobial effect, compared to the delivery ofthe agent in the absence of the nanoemulsions of the present invention.However, in some embodiments, the nanoemulsions are used without anotherantimicrobial agent (i.e., the nanoemulsion itself is the onlyantimicrobial portion of the composition).

There are an enormous amount of antimicrobial agents currently availablefor use in treating bacterial, fungal and viral infections. For acomprehensive treatise on the general classes of such drugs and theirmechanisms of action, the skilled artisan is referred to Goodman &Gilman's “The Pharmacological Basis of Therapeutics” Eds. Hardman etal., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996,(herein incorporated by reference in its entirety). Generally, theseagents include agents that inhibit cell wall synthesis (e.g.,penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); andthe imidazole antifungal agents (e.g., miconazole, ketoconazole andclotrimazole); agents that act directly to disrupt the cell membrane ofthe microorganism (e.g., detergents such as polmyxin and colistimethateand the antifungals nystatin and amphotericin B); agents that affect theribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol,the tetracyclines, erthromycin and clindamycin); agents that alterprotein synthesis and lead to cell death (e.g., aminoglycosides); agentsthat affect nucleic acid metabolism (e.g., the rifamycins and thequinolones); the antimetabolites (e.g., trimethoprim and sulfonamides);and the nucleic acid analogues such as zidovudine, gangcyclovir,vidarabine, and acyclovir which act to inhibit viral enzymes essentialfor DNA synthesis. Various combinations of antimicrobials may beemployed.

Actual amounts of compositions and any enhancing agents in thecompositions may be varied so as to obtain amounts of emulsion andenhancing agents at the site of treatment that are effective in killingvegetative as well as sporular microorganisms and neutralizing theirtoxic products. Accordingly, the selected amounts will depend on thenature and site for treatment, the desired response, the desiredduration of biocidal action and other factors. Generally, the emulsioncompositions of the invention will comprise at least 0.001% to 100%,preferably 0.01 to 90%, of emulsion per ml of liquid composition. It isenvisioned that viral infections may be treated using between about0.01% to 100% of emulsion per ml of liquid composition. Bacterialinfections may be attacked with compositions comprising between about0.001% to about 100% of emulsion per ml of liquid composition. Sporescan be killed by emulsions comprising from about 0.001% to about 100% ofemulsion per ml of liquid composition. These are merely exemplaryranges. It is envisioned that the formulations may comprise about0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about0.025%, about 0.05%, about 0.075%, about 0.1%, about 0.25%, about 0.5%,about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95% or about 100% of emulsion per ml ofliquid composition. It should be understood that a range between any twofigures listed above is specifically contemplated to be encompassedwithin the metes and bounds of the present invention. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated.

The person responsible for administration will, in any event, determinethe appropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by the FDA Office ofBiologics standards.

B. Decontamination and Sterilization

In general, the present invention contemplates compositions and methodsthat find use as environmental decontamination agents and for treatmentof casualties in both military and terrorist attack. The inactivation ofa broad range of pathogens, including vegetative bacteria and envelopedviruses (See e.g., Chatlyyne et al., “A lipid emulsion with effectivevirucidal activity against HIV-1 and other common viruses,” Foundationfor Retrovirology and Humana Health, 3rd Conference on retroviruses andOpportunistic Infections, Washington, D.C., U.S.A. [1996]) and bacterialspores, combined with low toxicity in experimental animals, makes thepresent emulsions suitable for use as general decontamination agentsbefore a specific pathogen is identified. Preferred compositions of thepresent invention can be rapidly produced in large quantities and arestable for many months at a broad range of temperatures. Theseproperties provide a flexibility that is useful for a broad range ofdecontamination applications.

For example, certain formulations of the present invention areespecially effective at destroying many of the bacterial spores andagents used in biological warfare. In this regard, the compositions andmethods of the present are useful in decontaminating personnel andmaterials contaminated by biological warfare agents. Solutions ofpresent compositions may be sprayed directly onto contaminated materialsor personnel from ground based, or aerial spraying systems. In certainof these applications, the present invention contemplates that aneffective amount of composition be contacted to contaminated materialsor personnel such that decontamination occurs. Alternatively, personaldecontamination kits can be supplied to military or civilians likely tobecome contaminated with biological agents.

The inactivation of a broad range of pathogens, including vegetativebacteria and enveloped viruses (See e.g., Chatlyyne et al., “A lipidemulsion with effective virucidal activity against HIV-1 and othercommon viruses,” Foundation for Retrovirology and Humana Health, 3rdConference on retroviruses and Opportunistic Infections, Washington,D.C., U.S.A. [1996]) and bacterial spores (See e.g., Hamouda et al., J.Infect. Disease 180:1939 [1999]), combined with low toxicity makes thepresent compositions particularly well suited for use as generaldecontamination agents before a specific pathogen is identified.

Thus, certain embodiments of the present invention specificallycontemplate the use of the present compositions in disinfectants anddetergents to decontaminate soil, machinery, vehicles and otherequipment, and waterways that may have been subject to an undesiredpathogen. Such decontamination procedures may involve simple applicationof the formulation in the form of a liquid spray or may require a morerigorous regimen. Also, the present emulsions can be used to treat cropsfor various plant viruses (in place of or for use with conventionalantibiotics). Nanoemulsions may also be used to decontaminate farmanimals, animal pens, surrounding surfaces, and animal carcasess toeliminate, for example, nonenveloped virus of hoof and mouth disease.

In addition to their use in decontamination of land and equipment, theformulations also find use in household detergents for generaldisinfectant purposes. Moreover, some embodiments of the presentinvention can be used to prevent contamination of food with bacteria orfungi (e.g., non-toxic compositions). This can be done either in thefood preparation process, or by addition to the food as an additive,disinfectant, or preservative.

The inventive emulsions are preferably used on hard surfaces in liquidform. Accordingly, the foregoing components are admixed with one or moreaqueous carrier liquids. The choice of aqueous carrier is not critical.However, it should be safe and it should be chemically compatible withthe inventive emulsions. In some embodiments, the aqueous carrier liquidcomprises solvents commonly used in hard surface cleaning compositions.Such solvents should be compatible with the inventive emulsions andshould be chemically stable at the pH of the emulsions. They should alsohave good filming/residue properties. Solvents for use in hard surfacecleaners are described, for example, in U.S. Pat. No. 5,108,660, hereinincorporated by reference in its entirety.

In preferred embodiments, the aqueous carrier is water or a misciblemixture of alcohol and water. The alcohol can be used to adjust theviscosity of the compositions. In some embodiments, the alcohols arepreferably C2-C4 alcohols. In particularly preferred embodiments,ethanol is employed. For example, in one preferred embodiment, theaqueous carrier liquid is water or a water-ethanol mixture containingfrom about 0 to about 50% ethanol. The present invention also embodiesnon-liquid compositions. These non-liquid compositions can be ingranular, powder or gel forms, preferably in granular forms.

Optionally, some compositions contain auxiliary materials that augmentcleaning and aesthetics so long as they do not interfere with theactivity of the inventive emulsions. The compositions can optionallycomprise a non-interfering auxiliary surfactant. A wide variety oforganic, water-soluble surfactants can optionally be employed. Thechoice of auxiliary surfactant depends on the desires of the user withregard to the intended purpose of the compositions and the commercialavailability of the surfactant. Other optional additives such asperfumes, brighteners, enzymes, colorants, and the like can be employedin the compositions to enhance aesthetics and/or cleaning performance.Detergent builders can also be employed in the compositions. Detergentbuilders sequester calcium and magnesium hardness ions that mightotherwise bind with and render less effective the auxiliary surfactantsor co-surfactants. Detergent builders are especially useful whenauxiliary surfactants or co-surfactants are employed, and are even moreuseful when the compositions are diluted prior to use with exceptionallyhard tap water e.g., above about 12 grains/gallon.

In other embodiments, the composition further comprise, sudssuppressors. In these embodiments, the compositions preferably comprisea sufficient amount of a suds suppressor to prevent excessive sudsingwhen contacting the compositions to hard surfaces. Suds suppressors areespecially useful in formulations for no-rinse application of thecomposition. The suds suppressor can be provided by known andconventional means. Selection of the suds suppressor depends on itsability to formulate in the compositions, and the residue and cleaningprofile of the compositions. The suds suppressor must be chemicallycompatible with the components in the compositions, it must befunctional at the pH range described herein, and it should not leave avisible residue on cleaned surfaces. Low-foaming co-surfactants can beused as suds suppressor to mediate the suds profile in the compositions.Co-surfactant concentrations between about 1 part and about 3% arenormally sufficient.

Examples of suitable co-surfactants for use herein include blockcopolymers (e.g., PLURONIC and TETRONIC gels [poly(ethyleneoxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) polymer gels, BASFCompany, Parispany, N.J.]) and alkylated (e.g.,ethoxylated/propoxylated) primary and secondary alcohols (e.g., TERIGTOL[Union Carbide, Danbury, Conn.]; POLY-TERGENTO [Olin Corporation,Norwalk, Conn.]). The optional suds suppressor preferably comprises asilicone-based material. These materials are effective as sudssuppressors at very low concentrations. At low concentrations, thesilicone-based suds suppressor is less likely to interfere with thecleaning performance of the compositions. An example of suitablesilicone-based suds suppressors for use in the compositions is DowCorning DSE. These optional but preferred silicone-based sudssuppressors can be incorporated into the composition by known andconventional means.

In still other embodiments, the compositions may be used by health careworkers, or any persons contacting persons or areas with microbialinfections, for their personal health-safety and decontamination needs.In addition, the inventive emulsions can be formulated into sprays forhospital and household uses such as cleaning and disinfecting medicaldevices and patient rooms, household appliances, kitchen and bathsurfaces, etc. In similar embodiments, the compositions may be used bysanitation and environmental services workers, food processing andagricultural workers and laboratory personnel when these individuals arelikely to contact infectious biological agents. Additionally, thecompositions may be used by travelers and persons contacting areaslikely to harbor infectious and pathological agents.

C. Food Preparation

The present invention also contemplates that certain compositionsdescribed herein may be employed in the food processing and preparationindustries in preventing and treating food contaminated with food bornbacteria, fungi and toxins. Thus, such compositions may be employed toreduce or inhibit microbial growth or otherwise abrogate the deleteriouseffects of microbial contamination of food. For these applications, theemulsion compositions are applied in food industry acceptable forms suchas additives, preservatives or seasonings.

The phrase “acceptable in the food industry” refers to compositions thatdo not substantially produce adverse, or allergic reactions when takenorally by humans or animals. As used herein, “acceptable in foodindustry media” includes any and all solvents, dispersion substances,any and all spices and herbs and their extracts. Except insofar as anyconventional additives, preservatives and seasonings are incompatiblewith the emulsions of the present invention, their use in preventing ortreating food born microbes and their toxic products is contemplated.Supplementary active ingredients may also be incorporated into thecompositions. For such applications, acceptable carriers may take theform of liquids, creams, foams, gels and may additionally comprisesolvents, emulsifiers, gelling agents, moisturizers, stabilizers,wetting agents, preservatives, sequestering agents, dyes, perfumes andother components commonly employed in food processing industry.

In another embodiment of the present invention, the compositions may bespecifically designed for applications such as disinfecting orsterilization food industry devices, equipment, and areas where food isprocessed, packaged and stored. For applications of this type, thecompositions may be conveniently provided in the form of a liquid orfoam, and may be provided with emulsifiers, surfactants, bufferingagents, wetting agents, preservatives, and other components commonlyfound in compositions of this type. In some embodiments, thecompositions are applied to produce or agricultural products prior to orduring transportation of those goods. Compositions of the invention maybe impregnated into absorptive materials commonly used in packagingmaterial for the prevention of food contamination during transport andstorage (e.g., cardboard or paper packaging). Other delivery systems ofthis type will be readily apparent to those skilled in the art.

Actual amounts of the emulsions and enhancing agents in the compositionsof the invention may be varied so as to obtain appropriateconcentrations of emulsion and enhancing agents to effectively preventor inhibit food contamination caused by food born microbes and theirtoxic products. Accordingly, the selected concentrations will depend onthe nature of the food product, packaging, storage procedure and otherfactors. Generally, the emulsion compositions of the invention willcomprise at least 0.001% to about 90% of emulsion in liquid composition.It is envisioned that the formulations may comprise about 0.001%, about0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%, about0.05%, about 0.075%, about 0.1% about 0.25%, about 0.5%, about 1.0%,about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 15180%, about85%, about 90%, about 95% or about 100% of emulsion per ml of liquidcomposition. It should be understood that a range between any twofigures listed above is specifically contemplated to be encompassedwithin the metes and bounds of the present invention.

In particular embodiments, emulsions can be used as disinfectants anddetergents to decontaminate and prevent microbial infection of food,soil and water, machinery and other equipment, and animals.

The inventive emulsions can be used by the food industry to preventcontamination. For example, inclusion of the emulsion within the foodproduct itself would be effective in killing bacteria that may have beenaccidentally contaminated meat or poultry. This could also allow theindustry to use a potentially broader spectrum of food products andreduce costs.

Certain embodiments of the present invention can also be used in thebeverage industry. For example, the inventive emulsions could beincluded in juice products to prevent growth of certain fungi, whichcause contamination and lead to production of mycotoxins, which aredangerous to consumers. Through the addition of small amounts of theinventive emulsions, the most common fungal contaminants in fruit juicewere prevented. This effect was achieved with as little as one part in10,000 of the emulsion (an amount which did not alter the flavor or thecomposition of the juice product). For example, in some embodiments,contamination of products such are GATORADE by organisms such asByssochlamys fulva are presented with the use of the nanoemulsions ofthe present invention.

The inventive emulsions can be used to essentially remove infectiousagents on machinery and other equipment. For example, the emulsions canbe used to eliminate contaminations in meat processing plants,particularly of organisms such as Listeria monocytogenes and Salmonellaemicroorganisms, by cleaning slaughterhouses or food packaging facilitieson a continual basis with the emulsion.

The person responsible for administration will, in any event, determinethe appropriate dose for individual application. Moreover, said aboveapplication should meet general safety and purity standards as requiredby the FDA office.

D. Kits

In other embodiments of the present invention, the methods andcompositions, or components of the methods and compositions may beformulated in a single formulation, or may be separated into separateformulations for later mixing during use, as may be desired for aparticular application. Such components may advantageously be placed inkits for use against microbial infections, decontaminating instrumentsand the like. In some embodiments, such kits contain all the essentialmaterials and reagents required for the delivery of the formulations ofthe present invention to the site of their intended action.

In some embodiments, intended for in vivo use, the methods andcompositions of the present invention may be formulated into a single orseparate pharmaceutically acceptable syringeable compositions. In thiscase, the container means may itself be an inhalant, syringe, pipette,eye dropper, or other like apparatus, from which the formulation may beapplied to an infected area of the body, such as the lungs, injectedinto an animal, or even applied to and mixed with the other componentsof the kit.

The kits of the present invention also typically include a means forcontaining the vials in close confinement for commercial sale (e.g.,injection or blow-molded plastic containers into which the desired vialsare retained). Irrespective of the number or type of containers, thekits of the invention also may comprise, or be packaged with, aninstrument for assisting with the injection/ad ministration or placementof the ultimate complex composition within the body of an animal. Suchan instrument may be an inhalant, syringe and antiseptic wipe, pipette,forceps, measured spoon, eyedropper or any such medically approveddelivery vehicle.

E. Modification, Preparation, and Delivery

The present invention further provides a variety of methods and systemsfor the modification of the nanoemulsions of the present invention, theincorporation of the nanoemulsions into other products, packaging anddelivery of the compositions of the present invention, and methods forreducing the costs associated with the use or handling of materials orsamples that might be contaminated with microorganisms. The followingdescription is intended to simply provide some examples of themodification, preparation, and delivery of the compositions of thepresent invention. Those skilled in the art will appreciate variationsof such methods.

In some embodiments, the present invention provides methods forimproving or altering the nanoemulsions described herein. Such methodsinclude, for example, taking a nanoemulsion described herein andchanging one or more components of the nanoemulsion. Such changesinclude, but are not limited to, adding or removing one or morecomponents. The altered nanoemulsion can then be tested to determine ifit has desired or useful properties. In some embodiments of the presentinvention, nanoemulsions of the present invention, or those derived fromthe nanoemulsions of the present invention are diluted. The dilutedsamples can then be tested to determine if they maintain the desiredfunctionality. In yet other embodiments of the present invention, thenanoemulsions of the present invention, or those derived from thenanoemulsions of the present invention are pass through a qualitycontrol (QC) and/or quality assurance (QA) procedure to confirm thesuitability of the nanoemulsion for sale or delivery to a user orretailer.

In some embodiments of the present invention, the nanoemulsions of thepresent invention are added to another product to add or improveanti-microbial capabilities of the product or to test a suspected orprovide a perceived improved anti-microbial capability to the product(i.e., it is contemplated that the addition of a nanoemulsion of thepresent invention into a product is within the scope of the presentinvention regardless of whether it has a detectable, or any,antimicrobial capabilities). For example, in some embodiments, thenanoemulsions of the present invention are added to cleaning ordisinfectant materials (e.g., household cleaning agents). In otherembodiments, the nanoemulsions are added to medical or first aidmaterials. For example, the nanoemulsions may be added to (or useddirectly as) sterilization agents and wound care products. In yet otherembodiments, the nanoemulsions are added to industrial products. Forexample, in some embodiments, the nanoemulsions are added to motor oilsto prevent or reduce, for example, fungal contamination. As describedabove, effective, stable emulsion can even be synthesized using motoroil as the oil component (e.g., W₂₀5GC Mobil 1). In still otherembodiments, the nanoemulsions are added to food products. For example,the nanoemulsions can be added to beverages to prevent the growth ofunwanted organisms in the beverage.

The nanoemulsion of the present invention, whether alone, or inconjunction with other materials can be provided in many different typesof containers and delivery systems. For example, in some embodiments ofthe present invention, the nanoemulsions are provided in a cream orother solid or semi-solid form. During the development of the presentinvention, it was determined that the emulsions of the present inventionmay be incorporated into hydrogel formulations while maintainingantimicrobial capabilities. The use of the emulsions in hydrogelprovides a number of useful features. For example, hydrogels can beprepared in semi-solid structures of desired sizes and shapes. Thisallows, for example, the insertion of the hydrogel materials into tubesor other passageways to create antimicrobrial filters (i.e., materialspassed through the hydrogel are decontaminated by the emulsions of thepresent invention).

The nanoemulsions can be delivered (e.g., to user or customers) in anysuitable container. Container can be used that provide one or moresingle use or multi-use dosages of the nanoemulsion for the desiredapplication. In some embodiments of the present invention, thenanoemulsions are provided in a suspension or liquid form. Suchnanoemulsions can be delivered in any suitable container including spraybottles (e.g., pressurized spray bottles). For industrial or otherlarge-scale uses, large volumes (e.g., tens to thousands of liters) ofnanoemulsion may be provided in a single container configuredappropriately to allow distribution or use of the nanoemulsion. Suchcontainers may be used in conjunction with large-scale manufacturingfacilities.

In some preferred embodiments of the present invention, nanoemulsions ofthe present invention are used in conjunction with an existing businesspractice to reduce the costs associated with or improve the safety ofthe operation of the business practice. For example, the use of thenanoemulsions of the present invention can reduce costs associated withthe use or handling of materials or samples that might be contaminatedwith microorganisms. In some embodiments, the nanoemulsions of thepresent invention are used to improve safety or reduce the costsassociated with the medical industries. For example, the nanoemulsionsfind use as cheap and efficient sterilization agents for use on medicalmaterials (e.g., surface that come in contact with animals, people, orbiological samples) or with patients (e.g., internally or externally).The nanoemulsions also find use as cheap and efficient sterilizationagents for food processing and handling and industrial applications. Insome such embodiments, the present invention provides non-toxicnanoemulsions. For example, nanoemulsions are provided herein thatinclude ingredients that are currently approved by the appropriateregulatory agencies (e.g., FDA, USDA, etc.) for use in medical,agriculture, and food applications. Furthermore, methods are providedherein for the generation of additional nanoemulsions with the desiredfunctionality that can be composed entirely of non-toxic and approvedsubstances. As such, the nanoemulsions of the present invention can beused in applications without incurring having to undergo the timeconsuming and expensive process of gaining regulatory approval. Indeed,the emulsions can be less toxic than the sum of their individualcomponents. For example, X8PC was tested to compare the lytic effect ofthe emulsion on sheep red blood cells tested on blood agar plates ascompared to the lytic effect of mixtures of the non-emulsifiedingredients. The data is present in FIG. 34. The two black bars in FIG.34 show the lytic effect of the X8PC nanoemulsion compared to the lyticeffect of a non-emulsified mixture of all the ingredients.

F. Additional Examples

The nanoemulsions of the present invention may be used alone, or inconjunction with other materials and products to perform specificdesired functions. For example, the nanoemulsions may be added to orformulated with health care products, hygiene products, and householdproducts to prevent contamination of the products and/or to add orimprove anti-microbial properties of the products. In some embodiments,the functional components of the products are included in the aqueousphase or oil phase of the nanoemulsion (e.g., any of the nanoemulsioncompositions described above). Components may be added prior to, during,or following emulsification.

Examples of formulations and uses include (ingredients andconcentrations are illustrative; modifications may be made asappropriate or desired): acne treatment (e.g., 0.10% adapalene, 20%azelaic acid, 2.5-20% benzoyl peroxide, 1% clindamycin, 1.5-2%erythromycin, 0.05% isotetrinoin, 1% meclocycline, 4% nicotinamide, 1-3%resorcinol, 0.5-5% salicylic acid, 0.5-5% sulfur, 6% sulfurated lime[dilute 1:10], 2.2 mg/ml tetracycline hydrochloride, and 0.025-0.1%tretinoin); deep pore purifying astringent (Witch Hazel); antacids(e.g., <600 mg/5 ml alumina [aluminum hydroxide], aluminum carbonate,aluminium phosphate, <850 mg/5 ml calcium carbonate, 540 mg/5 mlmagaldrate, <500 mg/5 ml magnesia (magnesium hydroxide), magnesiumcarbonate, magnesium oxide, magnesium trisilicate, sodium bicarbonate,<40 mg/5 ml simethicone); aphthous stomatitis treatment (e.g.,corticosteroids, 0.12% chlorhexidine); corticosteroids (e.g., 0.05%alclometasone dipropionat, 0.10% amcinonide, 0.025% beclomethasonedipropionate, 0.01-0.1% betamethasone and derivatives, 0.05% clobetasolpropionate and derivatives, 0.05% desonide, 0.25% desoximetasone, 0.10%dexamethasone and derivatives, 0.05% diforasone diacetate, 0.10%diflucortolone valerate, 0.03% flumethasone pivalate, 0.01-0.025%fluocinolone acetonide, 0.01-0-0.05% fluocinonide, 0.025-0.05%flurandrenolide, 0.005% fluticasone propionate, 0.10% halcinonide, 0.05%halobetasol propionate, 0.2-2.5% hydrocortisone derivatives, 0.10%mometasone furonate, 0.025-0.5% triamcinolone acetonide); insect bitetreatment/cold sore/local anesthetics (e.g., 5-20% benzocaine, 1%butamben, 0.50% dibucaine, 0.5-5% lidocaine, 1% pramoxine, 1% tetracine,+/−0.50% menthol); burn wound infections (e.g., 85 mg/gm mafenide, 1%silver sulfadiazine, 0.5-1.5% framycetin, 0.01% gramicidin [mixed withframycetin], 2% fusidic acid); calluses treatment (e.g., 2-20%resorcinol, resorcinol+sulfur 2%+5-8%); candidiasis (e.g., 2%butoconazole, 1% ciclopirox, 1-10% clotrimazole, clotrimazole andbetamethasone 1% and 0.05%, 150 mg/dose econazole, 2% ketoconozole, 500mg and 100,000 Units metronidazole and nystatin, 2-5% miconazole,100,000 Units/Gram nystatin, 100,000 Units and 1 mg/gram nystatin andtriamcinolone, 1% sulconazole, 0.4-0.8% terconazole, 6.50% tioconazole);antifungus products (e.g., 3% clioquinol, 1% haloprogin, 1% naftifine,1% tolnaftate, 1% terbinafine, 1% oxiconazole); Tinea versicolor (e.g.,1% haloprogin, 2% ketoconazole); ODOR GUARD Shoe Deodorizer (e.g., 5.0%Sodium chlorite); dandruff (e.g., 2% chloroxine, 1-25% coal tar, 2%ketoconazole, 1-2% pyrithione, 1-10% salicylic acid, 1-2.5% seleniumsulfide); dermatitis/psoriasis (e.g., corticosteroids); folliculitis(e.g., 3% clioquinol); herpes (e.g., 5% acyclovir); impetigo (2%mupirocin); insect repellent (e.g., 7.5-100% diethyltoluamide);moisturizing lotion (e.g., dimethicone, allantoin, camphor, menthol,eucalyptus); mouth infection (e.g., 0.12% chlorhexidine); pediculosiscapitis (e.g., 1% lindane [benzyl benzoate]); scabies (e.g., 0.50%malathion, 1-5% permethrin, 0.18-0.33% and 2.2-4% pyrethrins andpiperonyl butoxide); scabies (e.g., 10% crotamiton, 0.5-10% sulfur, 6%sulfurated lime); psoriasis (e.g., 0.1-1% anthralin, 0.01%calcipotriene, 1-25%, cool tar, 1% methoxsalen, 1-3% resorcinol);rosacea (e.g., 0.75% metronidazole, 2-10% sulfur); skin infection(Bacterial)/Ulcers (e.g., 1.0% chloramphenicol, 3.0% chlorotetracycline,1.0% clindamycin, 3.0% clioquinol, 1.5-2% erythromycin, 0.1% gentamycin,2-7% iodine, 2.0% mupirocin, 0.5% neomycin, 10,000 units/gm polymyxin B,500 units/gm bacitracin, 1.0% silver sulfadiazine, 3% tetracycline);spermicidal (e.g., nonoxynol 9, nanoxynol 9+/−condom); sunscreen agents(e.g., 5-15% aminobenzoic acid, 3% avobenzone, 3% dioxybenzone, 4-15%homosalate, 2-3% lisadimate, 3.5-5% menthyl anthranilate, 7-10%octocrylene, 2-7.5% octyl methoxycinnamate, 3-5% octyl salicylate, 2-6%oxybenzone, 1.4-8% padimate O, 1-4% phenylbenzimidazole sulfonic acid,1-5% roxadimate, 5-10% sulisobenzone, 2-25% titanium dioxide, 5-12%trolamine salicylate, zinc oxide); toothpaste (e.g., sodium fluoride,sodium monofluorophosphate, amine fluoride, stannous fluoride); teethwhiteners (e.g., hydrogen peroxide, carbopol 956, sodium hydroxide,sodium acid phosphate, sodium stannate); tarter fighting (e.g.,polypyrophosphate, tetrasodium pyrophosphate); toothache (e.g., 10-20%benzocaine); teeth sensitivity protection (e.g., baking soda, 5.0%potassium nitrate); mouthwash (e.g., 0.006% lysozyme, 0.006%lactoferrin, 4000 units/100 mL glucose oxidase, 4000 units/100 mLlactoperosidase); vaginosis (e.g., 2% clindamycin, 0.75-10%metronidazole); warts, common (e.g., 2-20% resorcinol, 13% or 40%salicylic acid); warts, flat (e.g., 0.025-0.1% tretinoin); eye drops(e.g., 70.0% dextran, 0.3% hydroxypropyl methylcellulose 2910, 1.4%polyvinyl alcohol, 0.6% povidone); contact lens cleaners (e.g., 3.0%hydrogen peroxide, citrate, tetronic 1304, AMP-95); contact lens (e.g.,sodium chloride, boric acid, sorbitol, edetate disodium); contact lensdisinfectant (e.g., 0.0010% polyquad [polyquaternium-1], 0.0005% aldox[myristamidopropyl dimethylamine]); deodorant (e.g., 19.0% aluminumzirconium); anti-bacterial deodorant soap (e.g., triclocarban); diaperRash (e.g., 40.0% zinc oxide, dimethicone); anti-bacterial wipes forpets (e.g., lidocaine HCl); cat litter (e.g., baking soda); dishwasherdetergent (e.g., 2.7% phosphorous, 1.19 g phosphates); tub and showercleaner (e.g., monocarbamide hydrochloride); glass and surface cleaner(e.g., 3.5% isopropanol, 0.3% propylene glycol); toilet bowl cleaner(e.g., 51.0% 1-Bromo-3-chloro-5,5-dimethylhydantonin, 23.3%1,3-dichloro-5,5-dimethylhydantonin, 9.0%1,3-dichloro-5-ethyl-5-methylhydantonin); laundry cleaner (e.g., 5.3%sodium nonanoxloxy benzene sulfonate, 5.3% perboric acid, sodium salt);and pet fresh-carpet cleaner (e.g., 0.3% geraniol, rosemary oil, cedaroil, geranium oil, citronella oil, lemongrass oil, cinnamon oil, mintoil).

Thus, in some embodiments, the present invention provides antimicrobialoil-in-water nanoemulsions having one or more of a first componentcomprising a solvent (e.g., ethanol, glycerol, polyethylene glycol,isopropanol), a second component comprising a halogen-containingcompound (e.g., benzethonium chloride, methylbenzethonium chloride,N-alkyldimethyl benzylammonium chloride),alkyldimethyl-3,4-dichlorobenzyl ammonium chloride, cetypyridiniumchloride), a third component comprising a surfactant (e.g., TWEEN-20,TRITON X-100, SDS, Poloxamer, sodium lauryl sulfate), and a fourthcomponent (e.g., an addition surfactant, methanol, EDTA, tributylphosphate, tyloxapol, 2-phenylphenol, sodium chloride, trisodium citratehydrate, citric acid, sodium fluoride, peppermint extract, NaOH,L-alanine, inosine, ammonium chloride, PBS, menthol, thymol, eucalyptol,methyl salicylate, triclosan, gycerin, natrosol, benzoyl peroxide,salicyclic acid, citrate buffer, sodium saccharin, tergitol,monoethanolamine, hypertonic saline solution, calcium chloride, hydrogenperoxide, polyamer 407).

V. Experimental Examples

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); μ (micron); M (Molar); μM(micromolar); mM (millimolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nM (nanomolar); ° C. (degrees Centigrade); and PBS(phosphate buffered saline).

Example 1 Methods of Formulating Emulsions

The emulsion is produced as follows: an oil phase is made by blendingorganic solvent, oil, and surfactant and then heating the resultingmixture at 37-90° C. for up to one hour. The emulsion is formed eitherwith a reciprocating syringe instrumentation or Silverson high sheermixer. The water phase is added to the oil phase and mixed for 1-30minutes, preferably for 5 minutes. For emulsions containing volatileingredients, the volatile ingredients are added along with the aqueousphase.

In a particular embodiment, the emulsion was formed as follows: an oilphase was made by blending tri-butyl phosphate, soybean oil, and asurfactant (e.g., TRITON X-100) and then heating the resulting mixtureat 86° C. for one hour. An emulsion was then produced by injecting waterinto the oil phase at a volume/volume ratio of one part oil phase tofour parts water. The emulsion can be produced manually, withreciprocating syringe instrumentation, or with batch or continuous flowinstrumentation. Methods of producing these emulsions are well known tothose of skill in the art and are described in e.g., U.S. Pat. Nos.5,103,497; and 4,895,452, (herein incorporated by reference in theirentireties). Table 2 shows the proportions of each component, the pH,and the size of the emulsion as measured on a Coulter LS 130 lasersizing instrument equipped with a circulating water bath.

TABLE 2 Per- centage Chemical of Each Mean Coulter Mean CoulterComponents Com- Size Range of Emulsion ponent pH (in Microns) (inMicrons) BCTP TRITON X-100   2% Tributyl phosphate   2% 5.16 1.0740.758-1.428 Oil (ex. Soybean)   16% Water   80% BCTP 0.1* TRITON X-1000.20% 5.37 0.944 0.625-1.333 Tributyl phosphate 0.20% Oil (ex. Soybean)1.60% Water   98% *This emulsion was obtained by diluting the BCTPemulsion with water in a ratio of 1:9

The emulsions of the present invention are highly stable. Indeed,emulsions were produced as described above and allowed to standovernight at room temperature in sealed 50 to 1000 mL polypropylenetubes. The emulsions were then monitored for signs of separation.Emulsions that showed no signs of separation were considered “stable.”Stable emulsions were then monitored over 1 year and were found tomaintain stability.

Emulsions were again produced as described above and allowed to standovernight at −20° C. in sealed 50 mL polypropylene tubes. The emulsionswere then monitored for signs of separation. Emulsions that showed nosigns of separation were considered “stable.” The BCTP and BCTP 0.1,emulsions have been found to be substantially unchanged after storage atroom temperature for at least 24 months.

Example 2 Characterization of an Exemplary Bacteria-InactivatingEmulsion of the Present Invention as an Emulsified Liposome Formed inLipid Droplets

A bacteria inactivating emulsion of the present invention, designatedX₈W₆₀PC, was formed by mixing a lipid-containing oil-in-water emulsionwith BCTP. In particular, a lipid-containing oil-in-water emulsionhaving glycerol monooleate (GMO) as the primary lipid andcetylpyridinium chloride (CPC) as a positive charge producing agent(referred to herein as GMO/CPC lipid emulsion or “W₈₀8P”) and BCTP weremixed in a 1:1 (volume to volume) ratio. U.S. Pat. No. 5,547,677 (hereinincorporated by reference in its entirety), describes the GMO/CPC lipidemulsion and other related lipid emulsions that may be combined withBCTP to provide the bacteria-inactivating oil-in-water emulsions of thepresent invention.

Example 3

In Vitro Bactericidal Efficacy Study I

Gram Positive Bacteria

In order to study the bactericidal efficacy of the emulsions of thepresent invention, the emulsions were mixed with various bacteria for 10minutes and then plated on standard microbiological media at varyingdilutions. Colony counts were then compared to untreated cultures todetermine the percent of bacteria killed by the treatment. Table 3summarizes the results of the experiment.

TABLE 3 Inoculum Emulsion Organism (CFU) % Killing Tested Vibriocholerae classical 1.3 × 10⁸ 100 BCTP Vibrio cholerae Eltor 5.1 × 10⁸100 BCTP Vibtio parahemolytica 4.0 × 10⁷ 98-100 BCTP

In order to study the bactericidal effect of the emulsions of thepresent invention on various vegetative forms of Bacillus species, anemulsion at three dilutions was mixed with four Bacillus species for 10minutes and then plated on microbiological medium. Colony counts werethen compared with untreated cultures to determine the percent ofbacteria killed by the treatment. Table 4 contains a summary of thebactericidal results from several experiments with the mean percentagekill in parenthesis.

TABLE 4 BCTP/ Dilution B. cerous B. circulans B. megaterium B. subtilus1:10  99% 95-99% 99% 99% (99%) (97%) (99%) (99%) 1:100  97-99% 74-93%96-97% 99% (98%) (84%) (96%) (99%) 1:1000  0% 45-60% 0-32% 0-39%  (0%)(52%) (16%) (20%)

Example 4 In Vitro Bactericidal Efficacy Study II Gram Negative Bacteria

To increase the uptake of the bacteria inactivating emulsions by thecell walls of Gram negative bacteria, thereby enhancing the microbicidaleffect of the emulsions on the resistant Gram negative bacteria, EDTA(ethylenediamine-tetraacetic acid) was premixed with the emulsions. TheEDTA was used in low concentration (50-25 μM) and the mix was incubatedwith the various Gram negative bacteria for 15 minutes. The microbicidaleffect of the mix was then measured on trypticase soy broth. The resultsare set forth in Table 5 below. There was over 99% reduction of thebacterial count using BCTP in 1/100 dilutions. This reduction of countwas not due to the killing effect of EDTA alone as shown from thecontrol group in which 250 μM of EDTA alone could not reduce thebacterial count in 15 minutes.

TABLE 5 Bacteria + Bacteria Bacteria + BCTP + Bacteria + alone BCTP EDTAEDTA Bacterium (CFU) (CFU) (CFU) (CFU) S. typhimunium 1,830,0001,370,000 40 790,000 S. dysenteriae 910,000 690,000 0 320,000

Example 5 In Vitro Bactericidal Efficacy Study III Vegetative and SporeForms

Bacillus cereus (B. cereus, ATCC #14579) was utilized as a model systemfor Bacillus anthracis. Experiments with BCTP diluted preparations tostudy the bactericidal effect of the compounds of the present inventionon the vegetative form (actively growing) of B. cereus were performed.Treatment in medium for 10 minutes at 37° C. was evaluated. Assummarized in Table 6, the BCTP emulsion is efficacious against thevegetative form of B. cereus. A 10 minute exposure with this preparationis sufficient for virtually complete killing of vegetative forms of B.cereus at all concentrations tested including dilutions as high as1:100.

TABLE 6 Emulsion Undiluted 1:10 1:100 BCTP >99% >99% 59->99% Avg = >99%Avg = >99% Avg = 82% Number of experiments = 4

The spore form of B. anthracis is one of the most likely organisms to beused as a biological weapon. Spores are well known to be highlyresistant to most disinfectants. As describe above, effective killing ofspores usually requires the use of toxic and irritating chemicals suchas formaldehyde or sodium hypochlorite (i.e., bleach). The sameexperiment was therefore performed with the spore form of B. cereus. Asshown in Table 7, treatment in both medium for 10 minutes at 37° C. wasnot sufficient to kill B. cereus spores.

TABLE 7 Emulsion Undiluted 1:10 1:100 BCTP 0%-12% 0% 0% Avg = 6% Avg =0% Avg = 0% Number of experiments = 2

To evaluate the efficacy of the compounds of the present invention onthe spore form of B. cereus over a period of time, BCTP was incorporatedinto solid agar medium at 1:100 dilution and the spores spread uniformlyon the surface and incubated for 96 hours at 37° C. No growth occurredon solid agar medium wherein BCTP had been incorporated, out to 96 hours(i.e., >99% killing, average>99% killing, 3 experiments).

In an attempt to more closely define the time at which killing of sporesby BCTP occurred, the following experiment was performed. Briefly, aspore preparation was treated with BCTP at a dilution of 1:100 andcompared to an untreated control. The number of colony forming units permilliliter (CFU/ml) was quantitated after 0.5, 1, 2, 4, 6, and 8 hours.As shown in FIG. 1, CFU/ml in the untreated control increased over thefirst 4 hours of incubation and then reached a plateau. Bacterial smearsprepared at time zero, 1, 2, 4 and 6 hours, and stained for sporestructures, revealed that by 2 hours no spore structures remained (FIGS.2A-2C). Thus, 100% germination of spores occurred in the untreatedcontrol by the 2 hour time point. In the spore preparation treated withBCTP, CFU/ml showed no increase over the first 2 hours and then declinedrapidly over the time period from 2-4 hours. The decline from baselineCFU/ml over 2-4 hours was approximately 1000-fold. Bacterial smearsprepared at the same time points and stained for spore structuresrevealed that spore structures remained to the end of the experiment at8 hours. Hence, germination of spores did not occur in the BCTP treatedculture due to either inhibition of the germination process or becausethe spores were damaged and unable to germinate. In order to determinewhether the emulsions were effective in killing other Bacillus speciesin addition to B. cereus, a similar experiment was performed asdescribed above, wherein spore preparations were treated with emulsionsand compared to an untreated control after four hours of incubation. Thefollowing Table 8 shows the results wherein the numbers represent themean sporicidal activity from several experiments.

TABLE 8 BCTP/ Dilution B. cereus B. circulans B. megaterium B. subtlius1:10 82% 61% 93% 31% 1:100 91% 80% 92% 39% 1:1000 47% 73% 94% 22%

Example 6 In Vivo Bactericidal Efficacy Study

Animal studies were preformed to demonstrate the protective andtherapeutic effect of the inventive emulsions in vivo. Bacillus cereusinfection in experimental animals has been used previously as a modelsystem for the study of anthrax (Burdon and Wende, 1960; Burdon et al.,1967; Lamanna and Jones, 1963). The disease syndrome induced in animalsexperimentally infected with B. cereusis in some respects similar toanthrax (Drobniewski, 1993; Fritz et al., 1995). The inventive emulsionswere mixed with B. cereus spores before injecting into mice.

Irrigation of Skin Wounds

A 1 cm skin wound was infected with 2.5×10⁷ B. cereus spores then closedwithout any further treatment. The other groups were infected with thesame number of spores. One hour later, the wounds were irrigated witheither inventive emulsion or saline to simulate post-exposuredecontamination. By 48 hours, there were large necrotic areassurrounding the wounds with an average area of 4.86 cm². In addition,60% of the animals in this group died as a result of the infection.Histology of these lesions indicated total necrosis of the dermis andsubdermis and large numbers of vegetative Bacillus organisms. Irrigationof experimentally infected wounds with saline did not result in anyapparent benefit.

Irrigation of wounds infected with B. cereus spores with inventiveemulsion showed substantial benefit, resulting in a consistent 98%reduction in the lesion size from 4.86 cm² to 0.06 cm². This reductionin lesion size was accompanied by a three-fold reduction in mortality(60% to 20%) when compared to experimental animals receiving either notreatment or saline irrigation. Histology of these lesions showed noevidence of vegetative Bacillus organisms and minimal disruption of theepidermis (Hamouda et al., 1999).

Subcutaneous Injection

CD-1 mice were injected with inventive emulsion diluted 1:10 in salineas a control and did not exhibit signs of distress or inflammatoryreaction, either in gross or histological analysis. To test thepathogenic effect of B. cereus spores in vivo and the sporicidal effectof inventive emulsion, a suspension of 4×10⁷ B. cereus spores was mixedwith saline or with inventive emulsion at a final dilution of 1:10 andthen immediately injected subcutaneously into the back of CD-1 mice.

Mice that were infected subcutaneously with B. cereus spores withoutinventive emulsion developed severe edema at 6-8 hours. This wasfollowed by a gray, necrotic area surrounding the injection site at18-24 hours, with severe sloughing of the skin present by 48 hours,leaving a dry, red-colored lesion.

Simultaneous injection of spores and inventive emulsion resulted in agreater than 98% 109 reduction in the size of the necrotic lesion from1.68 cm² to 0.02 cm² when the spores were premixed with inventiveemulsion. This was associated with minimal edema or inflammation(Hamouda et al., 1999).

Rabbit Cornea

The cornea of rabbits were irrigated with various concentrations of theinventive emulsions and monitored at 24 and 48 hours. No irritations orabnormalities were observed when compositions were used in therapeuticamounts.

Mucous Membrane

Intranasal toxicity was preformed in mice by installation of 25 μL of 4%of the nanoemulsion per nare. No clinical or histopathological changeswere observed in these mice.

Oral toxicity testing in rats was performed by gavaging up to 8 mL perkg of 25% nanoemulsion. The rats did not lose weight or show signs oftoxicity either clinically or histopathologically. There were noobserved changes in the gut bacterial flora as a result of oraladministration of the emulsions.

In a particular embodiment, Bacillus cereus was passed three times onblood agar (TSA with 5% sheep blood, REMEL). B. cereus was scraped fromthe third passage plate and resuspended in trypticase soy broth (TSB)(available from BBL). The B. cereus suspension was divided into twotubes. An equal volume of sterile saline was added to one tube and mixed0.1 cc of the B. cereus suspension/saline was injected subcutaneouslyinto 5 CD-1 mice. An equal volume of BCTP (diluted 1:5 in sterilesaline) was added to one tube and mixed, giving a final dilution of BCTPat 1:10. The B. cereus suspension/BCTP was incubated at 37° C. for 10minutes while being mixed 0.1 cc of the B. cereus suspension/BCTP wasinjected subcutaneously into 5 CD-1 mice. Equal volumes of BCTP (diluted1:5 in sterile saline) and TSB were mixed, giving a final dilution ofBCTP at 1:10. 0.1 cc of the BCTP/TSB was injected subcutaneously into 5CD-1 mice.

The number of colony forming units (cfu) of B. cereus in the inoculawere quantitated as follows: 10-fold serial dilutions of the B. cereusand B. cereus/BCTP suspensions were made in distilled H₂0. Duplicateplates of TSA were inoculated from each dilution (10 μl per plate). TheTSA plates were incubated overnight at 37° C. Colony counts were madeand the number of cfu/cc was calculated. Necrotic lesions appears to besmaller in mice which were inoculated with B. cereus which waspretreated with BCTP. The following Table 9 shows the results of theexperiment.

TABLE 9 Observation Inoculum ID# (24 hours) B. cereus 1528 necrosis atinjection 3.1 × 10⁷ site cfu/mouse 1529 necrosis at injection site 1530dead 1531 dead 1532 necrosis at injection site B. cereus 1348 necrosisat injection 8.0 × 10⁵ site cfu/mouse 1349 no reaction (BCTP treated)1360 no reaction 1526 necrosis at injection site 1527 necrosis atinjection site BCTP/TSB 1326 no reaction 1400 no reaction 1375 noreaction 1346 no reaction 1347 no reaction

Bacillus cereus was grown on Nutrient Agar (Difco) with 0.1% YeastExtract (Difco) and 50 μg/ml MnSO₄ for induction of spore formation. Theplate was scraped and suspended in sterile 50% ethanol and incubated atroom temperature for 2 hours with agitation in order to lyse remainingvegetative bacteria. The suspension was centrifuged at 2,500×g for 20minutes and the supernatant discarded. The pellet was resuspended indiH₂O, centrifuged at 2,500×g for 20 minutes, and the supernatantdiscarded. The spore suspension was divided. The pellet was resuspendedin TSB. 0.1 cc of the B. cereus spore suspension diluted 1:2 with salinewas injected subcutaneously into 3 CD-1 mice. Equal volumes of BCTP(diluted 1:5 in sterile saline) and B. cereus spore suspension weremixed, giving a final dilution of BCTP at 1:10 (preincubation time). 0.1cc of the BCTP/B. cereus spore suspension was injected subcutaneouslyinto 3 CD-1 mice. The number of colony forming units (cfu) of B. cereusin the inoculum was quantitated as follows. 10-fold serial dilutions ofthe B. cereus and B. cereus/BCTP suspensions were made in distilled H₂O.Duplicate plates of TSA were inoculated from each dilution (10 μl perplate). The TSA plates were incubated overnight at 37° C. Colony countswere made and the number of cfu/cc was calculated. Necrotic lesionsappeared to be smaller in mice which were inoculated with B. cereusspores which were pretreated with BCTP. The observations from thesestudies are shown in Table 10.

TABLE 10 Inoculum Observation (24 hours) B. cereus 2/3 (66%) miceexhibited necrosis at injection site 6.4 × 10⁶ spores/mouse B. cereus1/3 (33%) mice exhibited necrosis at injection site 4.8 × 10⁶spores/mouse (BCTP treated) B. cereus 3/3 (100%) mice exhibited necrosisat injection site 4.8 × 10⁶ vegetative forms/mouse Lysed B. cereus 3/3(100%) mice did not exhibit symptoms 4.8 × 10⁶ cfu/mouse BCTP/TSB 1/3(33%) mice appeared to have some skin necrosis

Bacillus cereus was grown on Nutrient Agar (Difco) with 0.1% YeastExtract (Difco) and 50 (g/ml MnSO₄ for induction of spore formation).The plate was scraped and suspended in sterile 50% ethanol and incubatedat room temperature for 2 hours with agitation in order J to lyseremaining vegetative bacteria. The suspension was centrifuged at 2,500×gfor 20 minutes and the supernatant discarded. The pellet was resuspendedin distilled H₂O, centrifuged at 2,500×g for 20 minutes, and thesupernatant discarded. The pellet was resuspended in TSB. The B. cereusspore suspension was divided into, three tubes. An equal volume ofsterile saline was added to one tube and mixed. 0.1 cc of the B. cereussuspension/saline was injected subcutaneously into 10 CD-1 mice. Anequal volume of BCTP (diluted 1:5 in sterile saline) was added to thesecond tube and mixed, giving a final dilution of BCTP at 1:10. The B.cereus spore suspension/BCTP (1:10) was incubated at 37° C. for 4 hourswhile being mixed, 0.1 cc of the B. cereus spore suspension/BCTP (1:110)was injected subcutaneously into 10 CD-1 mice. An equal volume of BCTP(diluted 1:50 in sterile saline) was added to the third tube and mixed,giving a final dilution of BCTP at 1:100. The B. cereus sporesuspension/BCTP (1:100) was incubated at 37° C. for 4 hours while beingmixed. 0.1 cc of the B. cereus spore suspension/BCTP (1:100) wasinjected subcutaneously into 10 CD-1 mice. Equal volumes of BCTP(diluted 1:5 in sterile saline) and TSB were mixed, giving a finaldilution of BCTP at 1:10. 0.1 cc of the BCTPFTSB was injectedsubcutaneously into 10 CD-1 mice. Equal volumes of BCTP (diluted 1:50 insterile saline) and TSB were mixed, giving a final dilution of BCTP at1:100. 0.1 cc of the BCTP/TSB was injected subcutaneously into 10 CD-1mice. The observations form these studies are shown in Table 11 andTable 12.

TABLE 11 Inoculum sc ID# Observation at 24 hours B. cereus 1 2.4 cm²skin lesion with 0.08 cm² 5.5 × 10⁷ necrotic area Spores/mouse 2 noabnormalities observed No treatment group 3 Moribund with 8 cm² skinlesion and hind 4 limb paralysis 5 3.52 cm² skin lesion 6 1.44 cm² skinlesion 7 3.4 cm² skin lesion 8 5.5 cm² skin lesion 9 5.5 cm² skin lesion10 3.3 cm² skin lesion with 0.72 cm² necrotic area 2.64 cm² skin lesionwith two necrotic areas (0.33 cm² and 0.1 cm²) Mean lesion size in Sporegroup alone 3.97 cm² (1/10 (10%) with no abnormalities observed) Note:Skin lesions grey in color with edema, necrotic areas red/dry.

TABLE 12 Inoculum sc ID # Observation at 24 hours B. cereus 41 noabnormalities observed 2.8 × 10⁷ 42 no abnormalities observedspores/mouse 43 1.2 cm² white skin lesion with grey center, in theslight edema BCTP 1:10 44 0.78 cm² white skin lesion treated group 450.13 cm² white skin lesion 46 2.2 cm² white skin lesion 47 1.8 cm² whiteskin lesion with 0.1 cm² brown area in center 48 1 cm² white skin lesionwith grey center 49 0.78 cm² white skin lesion 50 no abnormalitiesobserved Mean lesion size in BCTP 1:10 treatment group = 1.13 cm² (3/10(30%) with no abnormalities observed) B. cereus 51 2.1 cm² grey skinlesion 1.8 × 10⁷ 52 0.72 cm² grey skin lesion spores/mouse 53 1.5 cm²grey skin lesion in the 54 1.2 cm² grey skin lesion BCTP 1:100 55 3.15cm² grey skin lesion treated group 56 0.6 cm² grey skin lesion 57 0.5cm² grey skin lesion 58 2.25 cm² grey skin lesion 59 4.8 cm² grey skinlesion with necrotic area 1 cm diameter 60 2.7 cm² grey skin lesion Meanlesion size In BCTP 1:100 treatment group = 1.9 cm² (0/10 (0%) with noabnormalities observed) BCTP 1:10 alone 11 2.6 cm² white area 12 0.15cm² white area 13 no abnormalities observed 14 0.15 cm² white area 150.35 cm² white area 16 no abnormalities observed 17 0.12 cm² white area18 no abnormalities observed 19 0.56 cm² white area 20 0.3 cm² whitearea Mean lesion size In BCTP 1:10 alone group = 0.60 cm² (3/10 (30%)with no abnormalities observed) BCTP 1:100 21-30 no abnormalitiesobserved alone Mean lesion size in BCTP 1:100 alone group = 0 cm² (10/10(100%) with no abnormalities observed) TSB 31-40 no abnormalitiesobserved alone Mean lesion size In the TSB alone group = 0 cm² (10/10(100%) with no abnormalities observed)

Re-isolation of B. cereus was attempted from skin lesions, blood, liver,and spleen (Table 13). Skin lesions were cleansed with betadine followedby 70% sterile isopropyl alcohol. An incision was made at the margin ofthe lesion and swabbed. The chest was cleansed with betadine followed by70% sterile isopropyl alcohol. Blood was drawn by cardiac puncture. Theabdomen was cleansed with betadine followed by 70% sterile isopropylalcohol. The skin and abdominal muscles were opened with separatesterile instruments. Samples of liver and spleen were removed usingseparate sterile instruments. Liver and spleen samples were passedbriefly through a flame and cut using sterile instruments. The freshlyexposed surface was used for culture. BHI agar (Difco) was inoculatedand incubated aerobically at 37° C. overnight.

TABLE 13 B. cereus Re-isolation Inoculum sc ID# Necrospy from site ofskin lesion B. cereus  3 24 hours skin lesion >300 cfu 5.5 × 10⁷  6 48hours skin lesion >300 cfu spores/mouse  7 48 hours skin lesion >300 cfuin the  8 72 hours skin lesion 100 cfu Untreated group  9 72 hours skinlesion 25 cfu 10 72 hours skin lesion 100  1 96 hours skin lesion >300cfu  4 96 hours skin lesion >300 cfu  5 96 hours skin lesion >300 cfuMean CFU In Untreated Spore group = 214* *(6/9 (67%) >300 CFU) B. cereus48 48 hours skin lesion 17 cfu 2.8 × 10⁷ 50 48 hours skin lesion >300cfu spores/mouse 46 72 hours skin lesion >200 cfu in the 47 72 hoursskin lesion 100 cfu BCTP 1:10 49 72 hours skin lesion >300 cfu treatedgroup 41 96 hours skin lesion >300 cfu  42* 96 hours skin lesion 20 cfu43 cultures not done 44 96 hours skin lesion >300 cfu 45 cultures notdone 46 cultures not done Mean CFU in BCTP 1:10 group = 192* *(318(38%) >300 CFU) B. cereus 48 48 hours skin lesion 18 cfu 1.8 × 10⁷  50*48 hours skin lesion >300 cfu spores/mouse 52 72 hours skin lesion I cfuin the 54 72 hours re-isolation negative BCTP 1:100 56 72 hours skinlesion >300 cfu treated group 58 96 hours skin lesion 173 cfu 59 96hours skin lesion 4 cfu 60 96 hours skin lesion 6 cfu Mean CFU in BCTP1:100 group = 100 *(2/8 (25%) >00 CFU) *Although no lesions were presentin these mice, organisms were removed from the injection site.

Pretreatment of both vegetative B. cereus and B. cereus spores reducetheir ability to cause disease symptoms when introduced intoexperimental animals. This is reflected in the smaller size of skinlesions and the generally lower numbers of B. cereus recovered from thelesions. In addition, less frequent re-isolation of B. cereus fromblood, liver, and spleen occurs suggesting that septicemia may bepreventable.

Example 7 In Vivo Toxicity Study I

CD-1 mice were injected subcutaneously with 0.1 cc of the compounds ofthe present invention and observed for 4 days for signs of inflammationand/or necrosis. Dilutions of the compounds were made in sterile saline.Tissue samples from mice were preserved in 10% neutral buffered formalinfor histopathologic examination. Samples of skin and muscle (from micewhich were injected with undiluted compounds) sent for histologicalreview were reported to show indications of tissue necrosis. Tissuesamples from mice which were injected with diluted compounds were nothistologically examined. Tables 14 and 15 show the results of twoindividual experiments.

TABLE 14 Compound Mouse ID # Dilution Observation BCTP 1326 undilutednecrosis 1327 undiluted no reaction 1328 1:10 no reaction 1329 1:10 noreaction 1324 1:100 no reaction 1331 1:100 no reaction Saline 1344 noreaction 1345 no reaction

TABLE 15 Compound Mouse ID # Dilution Observation BCTP 1376 undilutednecrosis 1377 undiluted minimal necrosis 1378 1:10 no reaction 1379 1:10no reaction 1380 1:100 no reaction 1381 1:100 no reaction Saline 1394 noreaction 1395 no reaction

Guinea pigs were injected intramuscularly (in both hind legs) with 1.0cc of compounds of the present invention per site and observed for 4days for signs of inflammation and/or necrosis. Dilutions of thecompounds were made in sterile saline.

Tissue samples from guinea pigs were preserved in 10% neutral bufferedformalin for histological examination. Tissue samples were nothistologically examined.

TABLE 16 Compound Guinea Pig Dilution Observation BCTP 1023-1 undilutedno reaction 1023-2 1:10 no reaction 1023-3 1:100 no reaction Saline 1023-10 no reaction

The results of In Vivo Toxicity Study I show that subcutaneous andintramuscular injection of the compounds tested did not result ingrossly observable tissue damage and did not appear to cause distress inthe experimental animals (Table 16).

Example 8 In Vivo Toxicity Study II

One group of Sprague-Dawley rats each consisting of five males and fivefemales were placed in individual cages and acclimated for five daysbefore dosing. Rats were dosed daily for 14 days. On day 0-13, for 14consecutive days each rat in Group 1 received by gavage threemilliliters of BCTP, 1:100 concentration, respectively. Thethree-milliliter volume was determined to be the maximum allowable oraldose for rats. Prior to dosing on Day 0 and Day 7, each rat was weighed.Thereafter rats were weighed weekly for the duration of the study.Animals were observed daily for sickness or mortality. Animals wereallowed to rest for 14 days. On Day 28 the rats were weighed andeuthanized. The mean weight results of the oral toxicity study are shownin Table 17. Mean weights for males and females on Days 0, 7, and 14, 21and 28 and the mean weight gains from Day O— Day 28, are also shown inTable 17. One rat died due to mechanical trauma from manipulation of thegavage tubing during dosing on Day 14. All surviving rats gained weightover the 28 day course of the study and there was no illness reported.Thus, although tributyl phosphate alone is known to be toxic andirritating to mucous membranes, when incorporated into the emulsions ofthe present invention, these characteristics are not in evidence. TheBCTP emulsion, 1:100 concentration, was also tested for dermal toxicityin rabbits according to the protocols provided in 16 CFR § 1500.3. Theemulsion was not irritating to skin in the animals tested.

TABLE 17 Dose Body Body Body Body Body Weight Gain (g) Rat Volume Weight(g) Weight (g) Weight (g) Weight (g) Weight (g) Day 0 Number Sex mL Day0 Day 7 Day 14 Day 21 Day 28 Day 28 9028 m 3 332.01 356.52 388.66 429.9394.07 62.06 9029 m 3 278.62 294.65 296.23 310.7 392.6 113.98 9030 m 3329.02 360.67 325.26 403.43 443.16 114.14 9031 m 3 334.64 297.04 338.82357.5 416.89 82.25 9032 m 3 339.03 394.39 347.9 331.38 357.53 18.5 MEAN266.26 340.65 339.37 400.85 78.18 WTS 9063 F 3 302 298.08 388.66 338.41347.98 45.98 9064 F 3 254.54 247.97 256.78 278.17 279.2 24.66 9065 F 3225.99 253.81 273.38 290.54 308.68 82.69 9066 F 3 246.56 260.38 266.21235.12 272.6 26.04 9067 F 3 279.39 250.97 deceased MEAN 261.69 262.24296.25 285.56 302.11 53 WTS

General techniques for toxicity testing include dermal irritationtesting, eye irritation testing, subcutaneous test, intramuscular tests,open wound irrigation, intranasal tests, and oral tests. Dermal testscan be conducted on rabbits wherein 0.5 ml of 10% emulsion is applied tothe skin or rabbits for four hours. The skin reaction is recorded for upto 72 hours. A Draize scale is used to score the irritation. For eyeirritation testing, 0.1 ml of 10% emulsion is applied to the eye ofrabbits and the eye reaction is recorded for up to 72 hours. A Draizescale is used to score the irritation. Subcutaneous and intramusculartests inject 0.1 ml of 10% emulsion in mice. Two ml of 10% emulsion isapplied in an open wound irrigation test using mice. For intranasaltesting, 0.25 m/naris of 2-4% emulsion are applied to mice. For oraltesting, 4 ml/kg/day of 10% emulsion are given orally for 1 week or 8ml/kg of 100% emulsion is given in a single dose.

Example 9 In Vitro Study with Bacillus Anthracis

Experiments with X₈W₆₀PC preparations to study the bactericidal effectof the compounds of the present invention on the spore form of B.anthracis were performed. The sporicidal activity of different dilutionsof X₈W₆₀PC (in water) on six different strains of B. anthracis is shownin FIG. 3. As shown in FIGS. 4 and 5, X₈W₆₀PC killed over 98% of sevendifferent strains of anthrax (those of FIG. 3 and Ames, USAMRID) within4 hours and is as efficient as 1-10% bleach. Similar sporicidal activityis found with different dilutions of X₈W₆₀PC in media (FIG. 6). FIG. 7shows the time course for the sporicidal activity of X₈W₆₀PC against theDel Rio, Tex. strain of B. anthracis compared with zero time at roomtemperature. As shown, X₈W₆₀PC can kill anthrax spores in as little as30 minutes.

Example 10 Mechanisms of Action

The following Example provides an insight into a proposed the mechanismsof action of the emulsions of the present invention and to show theirsporicidal activity. This mechanism is not intended to limit the scopeof the invention an understanding of the mechanism is not necessary topractice the present invention, and the present invention is not limitedto any particular mechanism. The effect of a GMO/CPC lipid emulsion(“W₈₀8P”) and BCTP on E. coli was examined. W₈₀8P killed the E. coli (indeionized H₂O) but BCTP was ineffective against this organism. FIG. 8shows the control and FIG. 9 shows the E. coli treated with BCTP. Asshown in FIG. 9, the BCTP treated E. coli look normal, with definedstructure and intact lipid membranes. FIG. 10 shows the P10 treated E.coli, wherein the bacteria have vacuoles inside and the contents haveswollen so that the defined structure of the organism is lost. Withoutbeing bound to a particular theory (an understanding of the mechanism isnot necessary to practice the present invention, and the presentinvention is not limited to any particular mechanism), this observationsuggests that W₈₀8P kills the bacteria without lysing them and insteadcauses a change in the internal structure, evident by the vacuolizationand swelling. A second study was performed with Vibrio cholerae. DespiteVibrio cholerae being closely related to E. coli, both the BCTP, W₈₀8Pand X₈W₆₀PC killed this organism. Compared to the controlelectromicrograph (FIG. 11), the W₈₀8P treated Vibrio cholerae (FIG. 12)again shows swelling and changes in the interior of the organism, butthe cells remain intact. In contrast, the BCTP treated Vibrio cholerae(FIG. 13) are completely lysed with only cellular debris remaining.X₈W₆₀PC (FIG. 14) showed a combination of effects, where some of theorganisms are swelled but intact and some are lysed. This clearlysuggests that BCTP, W₈₀8P and X₈W₆₀PC work by different mechanisms. Athird comparative study was performed to evaluate efficacy of theemulsions at various concentrations. As shown in Table 18, X₈W₆₀PC ismore effective as a biocide at lower concentrations (higher dilutions)in bacteria sensitive to either W₈₀8P or BCTP. In addition, six otherbacteria that are resistant to W₈₀8P and BCTP are all susceptible toX8W₆₀PC. This difference in activity is also seen when comparing W₈₀8Pand BCTP and X₈W₆₀PC in influenza infectivity assays. As shown in FIG.15, both BCTP and X₈W₆₀PC are effective at a 1:10 and 1:100 dilutionsand additionally, X₈W₆₀PC is effective at the lowest concentration,1:1,000 dilution. In contrast, W₈₀8P has little activity even at 1:10dilution, suggesting that it is not an effective treatment for thisenveloped organism. In addition, X₈W₆₀PC kills yeast species that arenot killed by either W₈₀8P or BCTP.

TABLE 18 Lowest Nanoemulsion Concentration Required to Achieve Over 90%Killing of Selected Microorganisms W₈₀8P BCTP X₈W₆₀PC BacteriaStreptococcus pyogenes No killing  10% 0.1% Streptococcus aglactiae  1%*  1% ND Streptococcus pneumonia 10%*   1% 0.1% Staphylococcus aureus Nokilling No killing 0.1% Neissetia gonorhoeae ND   1% 0.1% Haemophilusinfluenzae 10%   1% 0.1% Vibrio cholerae  1% 0.1% 0.1% E. coli Nokilling# No killing 0.1% Salmonella typhimurium No killing# No killing 10% Shigella dysenteriae No killing# No killing 0.1% Proteus mirabilisNo killing# No killing   1% Pseudomonas aeruginosa No killing No killing 10% Bacillus anthracis spores No killing @ 4 H 0.1% @ 4 H 0.1%-0.02% @4 H Bacillus cereus spores 10% @ 4 H   1% @ 4 H 0.1% @ 4 H Bacillussubtilus spores No killing @ 24 H No killing @ 24 H 0.1% @ 4 H Yersiniaenterocolitica ND ND 0.1% Yersinia pseudotuberculosis ND ND 0.1% FungiCandida albicans No Killing No Killing   1% (ATCC 90028) Candidatropicalis No Killing No Killing   1% Viruses Influenza A H2N2 NoKilling   1% 0.1% Influenza B/Hong Kong/ ND   1% ND 5/72 Vaccinia ND  1% % Herpes simplex type I ND   1% 0.1% Sendai ND   1% ND Sindbis ND  1% ND Adenovirus ND No Killing ND *Data for lower concentrations notavailable. #No killing except in deionized water. 10 ND = Notdetermined.

Example 11 Further Evidence of the Sporicidal Activity of theNanoemulsion Against Bacillus Species

The present Example provides the results of additional investigations ofthe ability of particular embodiments of the emulsions of the presentinvention to inactivate different Bacillus spores. The methods andresults from these studies are outlined below.

Surfactant lipid preparations: BCTP, a water-in-oil nanoemulsion, inwhich the oil phase was made from soybean oil, tri-n-butyl phosphate,and TRITON X-100 in 80% water. X₈W₆₀C was prepared by mixing equalvolumes of BCTP with W₈₀8P which is a liposome-like compound made ofglycerol monostearate, refined Soya sterols, TWEEN 60, soybean oil, acationic ion halogen-containing CPC and peppermint oil.

Spore preparation: For induction of spore formation, Bacillus cereus(ATTC 14579), B. circulans (ATC 4513), B. megaterium (ATCC 14581), andB. subtilis (ATCC 11774) were grown for a week at 37° C. on NAYEMn agar(Nutrient Agar with 0.1% Yeast Extract and 5 mg/l MnSO₄). The plateswere scraped and the bacteria/spores suspended in sterile 50% ethanoland incubated at room temperature (27° C.) for 2 hours with agitation inorder to lyse the remaining vegetative bacteria. The suspension wascentrifuged at 2,500×g for 20 minutes and the pellet washed twice incold diH₂O. The spore pellet was resuspended in trypticase soy broth(TSB) and used immediately for experiments. B. anthracis spores, Amesand Vollum 1 B strains, were kindly supplied by Dr. Bruce Ivins(USAMRIID, Fort Detrick, Frederick, Md.), and prepared as previouslydescribed (Ivins et al., 1995). Four other strains of anthrax werekindly provided by Dr. Martin Hugh-Jones (LSU, Baton Rouge, La.). Thesestrains represent isolates with high allelic dissimilarity from SouthAfrica; Mozambique; Bison, Canada; and Del Rio, Tex.

In vitro sporicidal assays: For assessment of sporicidal activity ofsolid medium, trypticase Soy Agar (TSA) was autoclaved and cooled to 55°C. The BCTP was added to the TSA at a 1:100 final dilution andcontinuously stirred while the plates were poured. The sporepreparations were serially diluted (ten-fold) and 10 μl aliquots wereplated in duplicate (highest inoculum was 105 spores per plate). Plateswere incubated for 48 hours aerobically at 37° C. and evaluated forgrowth.

For assessment of sporicidal activity in liquid medium, spores wereresuspended in TSB. 1 ml of spore suspension containing 2×10⁶ spores(final concentration 106 spores/ml) was mixed with 1 ml of BCTP orX₈W₆₀PC (at 2× final concentration in diH₂O) in a test tube. The tubeswere incubated in a tube rotator at 37° C. for four hours. Aftertreatment, the suspensions were diluted 10-fold in diH₂O. Duplicatealiquots (25 μl) from each dilution were streaked on TSA, incubatedovernight at 37° C., and then colonies were counted. Sporicidal activityexpressed as a percentage killing was calculated:

$\frac{{{cfu}\mspace{11mu}\lbrack{initial}\rbrack} - {{cfu}\mspace{11mu}\left\lbrack {{post}\text{-}{treatment}} \right\rbrack}}{{cfu}\;\lbrack{initial}\rbrack} \times 100.$

The experiments were repeated at least 3 times and the mean of thepercentage killing was calculated.

Electron microscopy: B. cereus spores were treated with BCTP at a 1:100final dilution in TSB using Erlenmeyer flasks in a 37° C. shakerincubator. Fifty ml samples were taken at intervals and centrifuged at2,500×g for 20 minutes and the supernatant discarded. The pellet wasfixed in 4% glutaraldehyde in 0.1 M cacodylate (pH 7.3). Spore pelletswere processed for transmission electron microscopy and thin sectionsexamined after staining with uranyl acetate and lead citrate.

Germination inhibitors/simulators: B. cereus spores (at a finalconcentration 10⁶ spores/ml) were suspended in TSB with either thegermination inhibitor D-alanine (at final concentration of 1 μM) or withthe germination stimulator L-alanine+inosine (at final concentration of50 μM each) (Titball and Manchee, 1987; Foster and Johnston., 1990;Shibata et al., 1976) and then immediately mixed with BCTP (at a finaldilution of 1:100) and incubated for variable interval. Then themixtures were serially diluted, plated and incubated overnight. The nextday the plates were counted and percentage sporicidal activity wascalculated.

In vivo sporicidal activity: Two animal models were developed; in thefirst B. cereus spores (suspended in sterile saline) were mixed with anequal volume of BCTP at a final dilution of 1:10. As a control, the sameB. cereus spore suspension was mixed with an equal volume of sterilesaline. 100 μl of the suspensions containing 4×10⁷ spores was thenimmediately injected subcutaneously into CD-1 mice.

In the second model, a simulated wound was created by making an incisionin the skin of the back of the mice. The skin was separated from theunderlying muscle by blunt dissection. The “pocket” was inoculated with200 μl containing 2.5×10⁷ spores (in saline) and closed using woundclips. One hour later, the clips were removed and the wound irrigatedwith either 2 ml of sterile saline or with 2 ml of BCTP (1:10 in sterilesaline). The wounds were then closed using wound clips. The animals wereobserved for clinical signs. Gross and histopathology were performedwhen the animals were euthanized 5 days later. The wound size wascalculated by the following formula: ½a×½b×π where a and b are twoperpendicular diameters of the wound.

In vitro sporicidal activity: To assess the sporicidal activity of BCTP,spores from four species of Bacillus genus, B. cereus, B. circulans, B.megatetium, and B. subtilis were tested. BCTP at 1:100 dilution showedover 91% sporicidal activity against B. cereus and B. megaterium in 4hours (FIG. 16). B. circulans was less sensitive to BCTP showing 80%reduction in spore count, while B. subtilis appeared resistant to BCTPin 4 hours. A comparison of the sporicidal effect of BCTP (at dilutionsof 1:10 and 1:100) on B. cereus spores was made with a 1:100 dilution ofbleach (i.e., 0.0525% sodium hypochlorite), and no significantdifference was apparent in either the rate or extent of sporicidaleffect. The other nanoemulsion, X₈W₆₀PC, was more efficient in killingthe Bacillus spores. At 1:1000 dilution, it showed 98% killing of B.cereus spores in 4 hours (compared to 47% with 1:1000 dilution of BCTP).X₈W₆₀PC at 1:1000 dilution resulted in 97.6% killing of B. subtilisspores in 4 hours, in contrast to its resistance to BCTP.

B. cereus sporicidal time course: A time course was performed to analyzethe sporicidal activity of BCTP diluted 1:100 and X₈W₆₀PC diluted 1:1000against B. cereus over an eight hour period. Incubation of BCTP diluted1:100 with B. cereus spores resulted in a 77% reduction in the number ofviable spores in one hour and a 95% reduction after 4 hours. Again,X₈W₆₀PC diluted 1:1000 was more effective than BCTP 1:100 and resultedin about 95% reduction in count after 30 minutes (FIG. 17).

BCTP B. anthracis sporicidal activity: Following initial in vitroexperiments, BCTP sporicidal activity was tested against two virulentstrains of B. anthracis (Ames and Vollum 1B). It was found that BCTP ata 1:100 final dilution incorporated into growth medium completelyinhibited the growth of 1×10⁵ B. anthracis spores. Also, 4 hoursincubation with BCTP at dilutions up to 1:1000 with either the Ames orthe Vollum 1 B spores resulted in over 91% sporicidal activity when themixtures were incubated at RT, and over 96% sporicidal activity when themixtures were incubated at 37° C. (Table 19).

TABLE 19 BCTP sporicidal activity against 2 different strains ofBacillus anthracis spores as determined by colony reduction assay (%killing). BCTP at dilutions up to 1:1000 effectively killed >91% of bothspore strains in 4 hours at either 27 or 37° C.; conditions thatdiffered markedly in the extent of spore germination. Sporicidalactivity was consistent at spore concentrations up to 1 × 10⁶/ml. AmesAmes (cont) Vollum 1 B B. anthracis Room Temp. 37° C. Room Temp. 37° C.BCTP 1:10 91% 96% 97% 99% BCTP 1:100 93% 97% 97% 98% BCTP 1:1000 93% 97%98% 99%

X₈W₆₀PC B. anthracis sporicidal activity: Since X₈W₆₀PC was effective athigher dilutions and against more species of Bacillus spores than BCTP,it was tested against 4 different strains of B. anthracis at dilutionsup to 1:10,000 at RT to prevent germination. X₈W₆₀PC showed peak killingbetween 86% and 99.9% at 1:1000 dilution (Table 20).

TABLE 20 X₈W₆₀PC sporicidal activity against 4 different strains of B.anthracis representing different clinical isolates. The spores weretreated with X₈W₆₀PC at different dilutions in RT to reduce germination.There as no significant killing at low dilutions. The maximum sporicidaleffect was observed at 1:1000 dilution. South Bison, Del Rio, B.Anthracis Africa Canada Mozambigue Texas X₈W₆₀PC 1:10 81.8 85.9 41.9 38X₈W₆₀PC 1:100 84 88.9 96.5 91.3 X₈W₆₀PC 1:1000 98.4 91.1 99.9 86 X₈W₆₀PC1:5,000 79.7 41.3 95.7 97.1 X₈W₆₀PC 1:10,000 52.4 80 ND ND

Electron microscopy examination of the spores: Investigations werecarried out using B. cereus because it is the most closely related to B.anthracis. Transmission electron microscopy examination of the B. cereusspores treated with BCTP diluted 1:100 in TSB for four hours revealedphysical damage to the B. cereus spores, including extensive disruptionof the spore coat and cortex with distortion and loss of density in thecore (FIG. 18).

Germination stimulation and inhibition: To investigate the effect ofinitiation of germination on the sporicidal effect of BCTP on Bacillusspores, the germination inhibitors D-alanine (Titball and Manchee, 1987;Foster and Johnston, 1990), and germination simulators L-alanine andinosine (Shibata et al., 1976) were incubated with the spores and BCTPfor 1 hour. The sporicidal effect of BCTP was delayed in the presence of10 mM D-alanine and accelerated in the presence of 50 μM L-alanine and50 μM inosine (FIG. 19).

In vivo sporicidal activity: Bacillus cereus infection in experimentalanimals had been previously used as a model system for the study ofanthrax and causes an illness similar to experimental anthrax infection(Welkos et al., 1986; Drobniewski, 1993; Burdon and Wende, 1960; Burdonet al., 1967; Fritz et al., 1995 et al., 1995; Welkos and Friedlander,1988). Two animal models of cutaneous B. cereus disease were developedto assess the in vivo efficacy of BCTP. Because these models involvesubcutaneous administration of the nanoemulsion, in vivo toxicitytesting of BCTP was performed prior to this application. CD-1 miceinjected with BCTP diluted 1:10 in saline as a control did not exhibitsigns of distress or inflammatory reaction, either in gross orhistological analysis (FIG. 20A, FIG. 20B). To test the pathogeniceffect of B. cereus spores in vivo and the sporicidal effect of BCTP, asuspension of 4×10⁷ B. cereus spores was mixed with saline or with BCTPat a final dilution of 1:10 and then immediately injected subcutaneouslyinto the back of CD-1 mice. Mice which were infected subcutaneously withB. cereus spores without BCTP developed severe edema at 6-8 hours. Thiswas followed by a gray, necrotic area surrounding the injection site at18-24 hours, with severe sloughing of the skin present by 48 hours,leaving a dry, red-colored lesion (FIG. 20C, FIG. 20D). Simultaneousinjection of spores and BCTP resulted in a greater than 98% reduction inthe size of the necrotic lesion from 1.68 cm² to 0.02 cm² when thespores were premixed with BCTP. This was associated with minimal edemaor inflammation (FIG. 20E, FIG. 20F).

In additional studies, a 1 cm skin wound was infected with 2.5×10⁷ B.cereus spores then closed without any further treatment (FIG. 21A, FIG.21B). The other groups were infected with the same number of spores,then 1 hour later the wounds were irrigated with either BCTP or salineto simulate post-exposure decontamination. Irrigation of experimentallyinfected wounds with saline did not result in any apparent benefit (FIG.21C, FIG. 21D). BCTP irrigation of wounds infected with B. cereus sporesshowed substantial benefit, resulting in a consistent 98% reduction inthe lesion size from 4.86 cm² to 0.06 cm² (FIG. 21E, FIG. 21F). Thisreduction in lesion size was accompanied by a four-fold reduction inmortality (80% to 20%) when compared to experimental animals receivingeither no treatment or saline irrigation.

Example 12 Effect of Surfactant Lipid Preparations (SLPS) on Influenza AVirus Infectivity In Vitro

Enveloped viruses are of great concern as pathogens. They spread rapidlyand are capable of surviving out of a host for extended periods.Influenza A virus was chosen because it is a well accepted model to testanti-viral agents (Karaivanova and Spiro, 1998; Mammen et al, 1995;Huang et al, 1991). Influenza is a clinically important respiratorypathogen that is highly contagious and responsible for severe pandemicdisease (Mulder and Hers, 1972).

The envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA)not only determine the antigenic specificity of influenza subtypes(Schulze, 1997), but they mutate readily and, as a result, may allow thevirus to evade host defense systems. This may result in the initiationof disease in individuals that are immune to closely related strains.The following is a description of the methods and composition used fordetermining the efficacy of SLPs in preventing influenza A infectivity.

Surfactant lipid preparations (SLPs): The SLPs were made in a two-stepprocedure. An oil phase was prepared by blending soybean oil withreagents listed in Table 1 and heating at 86° C. for one hour (Florence,1993). The SLPs were then formed by injecting water or 1% bismuth inwater (SS) into the oil phase at a volume/volume ratio using areciprocating syringe pump.

Viruses: Influenza virus A/AA/6/60 (Hedocher et al., 1996) was kindlyprovided by Dr. Hunein F. Maassab (School of Public Health, Universityof Michigan). Influenza A virus was propagated in the allantoic cavitiesof fertilized pathogen-free hen eggs (SPAFAS, Norwich, Conn.) usingstandard methods (Barrett and Inglis, 1985). Virus stock was kept inaliquots (10⁸ pfu/ml) of infectious allantoic fluids at −80° C.Adenoviral vector (AD.RSV ntlacZ) was provided by Vector Core Facility(University of Michigan Medical Center, Ann Arbor, Mich.) and was keptin aliquots (1012 pfu/ml at −80° C.). The vector is based on a humanadenoviral (serotype 5) genomic backbone deleted of the nucleotidesequence spanning E1A and E1B and a portion of E3 region. This impairsthe ability of the virus to replicate or transform nonpermissive cells.It carries the Eschetichia coli LacZ gene, encoding, β-galactosidase,under control of the promoter from the Rouse sarcoma virus long terminalrepeat (RSV-LTR). It contains a nuclear targeting (designated as nt)epitope linked to the 5′ end of the LacZ gene to facilitate thedetection of protein expression (Baragi et al., 1995).

Cells: Madin Darby Canine Kidney (MDCK) cells were purchased from theAmerican Type Culture Collection (ATCC; Rockville, Md.) and 293 cells(CRL 1573; transformed primary embryonic human kidney) were obtainedfrom the Vector Core Facility (University of Michigan Medical Center,Ann Arbor, Mich.). The 293 cells express the transforming gene ofadenovirus 5 and therefore restore the ability of Ad.RSV ntlacZ vectorto replicate in the host cell (Graham et al., 1977).

Cell maintenance media: MDCK cells were maintained in Eagle's minimalessential medium with Earle's salts, 2 mM L-glutamine, and 1.5 g/lsodium bicarbonate (Mediatech, Inc., Herndon, Va.) containing 10% fetalbovine serum (FBS; Hyclone Laboratories, Logan, Utah). The medium wassupplemented with 0.1 mM non-essential amino acids, 1.0 mM sodiumpyruvate, 100 U penicillin/ml and streptomycin 100 μg/ml (LifeTechnologies, Gaithersburg, Md.). The 293 cells were maintained inDulbecco's modified Eagle medium (Mediatech, Inc., Herndon, Va.),containing 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1.0mM sodium pyruvate. It also contained 100 U penicillin/ml andstreptomycin 100 μg/ml (Life Technologies, Gaithersburg, Md.) and wassupplemented with 10% FBS (Hyclone Laboratories, Logan, Utah).

Virus infection media: Influenza A infection medium was the MDCK cellmaintenance medium (without FBS) supplemented with 3.0 μg/ml oftolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin(Worthington Biochemical Corporation, Lakewood, N.J.). Adenovirusinfection medium was 293) cell maintenance medium with a reducedconcentration of serum (2% FBS).

Influenza A overlay medium: Overlay medium consisted of equal amounts of2× infection medium and 1.6% SEAKEM ME agarose (FMC BioProducts,Rockland, Md.). Staining agarose overlay medium consisted of agaroseoverlay medium plus 0.01% neutral red solution (Life Technologies,Gaithersburg, Md.) without TPCK-treated trypsin.

Plaque reduction assays (PRA): The plaque reduction assay was performedwith a modification of the method described elsewhere (Hayden et al.,1980). MDCK cells were seeded at 1×10⁵ cells/well in 12-well FALCONplates and incubated at 37° C./5% CO₂ for 3 days. Approximately 1×10⁸pfu of influenza A virus was incubated with surfactant lipidpreparations as described below. The influenza A virus-SLP treatmentsand controls were diluted in infection medium to contain 30-100 pfu/250μl. Confluent cell monolayers were inoculated in triplicate on 3 platesand incubated at 37° C./5% CO₂ for 1 h. The inoculum/medium wasaspirated and 1 ml of agarose overlay medium/well was added and plateswere incubated at 37° C./5% CO₂ until plaques appeared. Monolayers werestained with the agarose overlay medium and incubation was continued at37° C./5% CO₂. Plaques were counted 6-12 h after staining. The averageplaque count from 9 wells with lipid preparation concentration wascompared with the average plaque count of untreated virus wells.

In situ cellular enzyme-linked immunosorbent assay (ELISA): To detectand quantitate viral proteins in MDCK cells infected with influenza Avirus, the in situ cellular ELISA was optimized. Briefly, 2×10⁴ MDCKcells in 100 μl complete medium were added to flat-bottom 96-wellmicrotitre plates and incubated overnight. On the next day, the culturemedium was removed and cells were washed with serum free maintenancemedium. One hundred μl of viral inoculum was added to the wells andincubated for 1 hour. The viral inoculum was removed and replaced with100 μl of MDCK cell maintained medium plus 2% FBS. The infected MDCKcells were incubated for an additional 24 h. Then the cells were washedonce with PBS and fixed with ice cold ethanol:acetone mixture (1:1) andstored at −20° C. On the day of the assay, the wells of fixed cells werewashed with PBS and blocked with 1% dry milk in PBS for 30 min. at 37°C. One hundred μl of ferret anti-influenza A virus polyclonal antibodyat 1:1000 dilution (kindly provided by Dr. Hunein F. Maassab, School ofPublic Health, University of Michigan) was added to the wells for 1 hrat 37° C. The cells were washed 4 times with washing buffer (PBS and0.05% TWEEN-20), and incubated with 100 μl at 1:1000 dilution of goatanti-ferret peroxidase conjugated antibody (Kirkegaard & PerryLaboratories, Gaithersburg, Mass.) for 30 min. at 37° C. Cells werewashed 4 times and incubated with 100 μl of 1-STEP TURBO TMB-ELISAsubstrate (Pierce, Rockford, Ill.) until color had developed. Thereaction was stopped with 1 N sulfuric acid and plates were read at awavelength of 450 nm in an ELISA microtiter reader.

β-galactosidase assay: β-galactosidase assay was performed on cellextracts as described elsewhere (Lim, 1989). Briefly, 293 cells wereseeded on 96-well “U”-bottom tissue culture plates at approximately4×10⁴ cells/well and incubated overnight at 37° C./5% CO₂ in maintenancemedium. The next day, the medium was removed and the cells were washedwith 100 μl Dulbecco's phosphate buffered saline (DPBS). Adenovirusstock was diluted in infection medium to a concentration of 5×10⁷ pfu/mland mixed with different concentrations of BCTP as described below.After treatment with BCTP, virus was diluted with infection medium to aconcentration of 1×10⁴ pfu/ml and overlaid on 293 cells. Cells wereincubated at 37° C./5% CO₂ for 5 days, after which the plates werecentrifuged, the medium was removed and the cells were washed threetimes with PBS without Ca++ and Mg++. After the third wash, the PBS wasaspirated and 100 μl of 1× Reporter Lysis Buffer (Promega, Madison,Wis.) was placed in each well. To enhance cell lysis, plates were frozenand thawed three times and the β-galactosidase assay was performedfollowing the instruction provided by the vendor of β-galactosidase(Promega, Madison, Wis.) with some modifications. Five microliters ofcell extract was transferred to a 96-well flat bottom plate and mixedwith 45 μl of 1× Reporter Lysis Buffer (1:10). Subsequently 50 μl of 2×assay buffer (120 mM Na₂HPO₄, 80 mM NaH₂PO₄, 2 mM MgCl₂, 100 mMβ-mercaptoethanol, 1.33 mg/ml ONPG (Sigma, St. Louis, Mo.) were addedand mixed with the cell extract. The plates were incubated at RT until afaint yellow color developed. At that time the reaction was stopped byadding 100 (1 of 1 M sodium bicarbonate. Plates were read at awavelength of 420 nm in an ELISA microplate reader. A standard,consisting of (u/μl β-galactosidase (Sigma, St. Louis, Mo.) supplementedin 50 mM bicine buffer (Sigma, St. Louis, Mo.), pH 7.5 and 100 (g/mlBSA) diluted in the 1× Reporter Lysis Buffer, was run with all assays.The units of β-galactosidase in each cell extract was calculated byregression analysis by reference to the levels in the standard anddivided by milligrams of protein in the cell extract sample.

Cellular toxicity and virus treatment with lipid preparations: Prior toviral susceptibility testing, cytotoxicity of SLPs on MDCK and 293)cells was assessed by microscope inspection and MTT assay. The dilutionsof the mixture of virus and SLPs applied in susceptibility testing weremade to be at least one order of magnitude higher than the safeconcentration of SLP assessed. Approximately 1×10⁸ pfu of eitherinfluenza A or adenovirus were incubated with lipid preparation at finalconcentrations of 1:10, 1:100, and 1:1000 for different time periods asindicated in results on a shaker. After incubation, serial dilutions ofthe SLP/virus mixture were made in proper infection media and overlaidon MDCK (influenza A) or 293 (adenovirus) cells to perform PRA, cellularELISA or β-galactosidase assays as described above.

Electron microscopy: Influenza A virus was semi-purified from allantoicfluid by passing through a 30% sucrose cushion prepared with GTNE(glycine 200 mM, Tris-HCl 10 mM (pH 8.8), NaCl 100 mM, and EDTA 1 mM)using ultra centrifugation (Beckman rotor SW 28 Ti, at 20,000 rpm for 16hours). Pelleted virus was reconstituted in GTNE. Ten microliters ofrespective samples (adenovirus, influenza virus, adenovirus+BCTP,influenza virus+BCTP) were incubated for 15 and 60 min, then placed onparlodian coated 200 mesh copper grids for 2 min. Then 5 μl of 2%cacodylated-buffered glutaraldehyde was added. The fluid was removedwith filter paper after 3 min. Ten microliters of 7% uranyl acetate wasadded to the grid and drawn off with filter paper after 30 sec. Thegrids were allowed to dry 10 min and examined on a Philips EM400Ttransmission electron microscope. Micrographs were recorded in Fuji FGfilm at magnifications of 200,000×.

Susceptibility testing of influenza A to SLPS: the effect of foursurfactant lipid preparations (BCTP, NN, W₈₀8P, and SS) on influenza Ainfection of MDCK cells was investigated. All tested preparationsinhibited influenza A virus infection to varying degrees as shown inFIG. 22. BCTP and SS exhibited over 95% inhibition of influenza Ainfection at a 1:10 dilution. NN and W₈₀8P showed only an intermediateeffect on influenza A virus, reducing infection by approximately 40%.BCTP's virucidal effect was undiminished even at a 1:100 dilution. SSshowed less effect at a 1:100 dilution inhibiting influenza A infectionby 55%. These two lipid preparations at 1:1000 dilution displayed onlyweak inhibitory effect on virus infectivity at the range of 22-29% (FIG.23B).

Since BCTP and SS both showed strong inhibitory effect on virusinfectivity, PRA was used to verify data obtained from cellular ELISA.PRA confirmed the efficacy of BCTP and SS. BCTP reduced the number ofplaques from an average of 50.88 to 0 at a 1:10 dilution (Table 21). Atdilution 1:100, BCTP maintained virucidal effectiveness. At dilution1:100 SS reduced the number of plaques only approximately 7% as comparedwith untreated virus.

TABLE 21 Plaque forming Plaque forming Treatment units units Dilution ofthe agent: BCTP SS 1:10^(a) 0.00^(b) (+/− 0.00)^(c) 0.00 (+/−0.00) 1:1000.00 (+/−0.00) 1.55 (+1-0.12) Untreated virus 50.88 (+/−1-0.25) 23.52(+/−0.18) ^(a)Virus was incubate with SLPs for 30 minutes. ^(b)Number ofplaques.

Kinetics of BCTP action on influenza A virus: To investigate the timerequirement for BCTP to act on influenza A infectivity, virus wasincubated with BCTP at two dilutions (1:10, 1:100) and four differenttime intervals (5, 10, 15, 30 min). Subsequently, plaque reduction assaywas performed. As shown in Table 22, after five min of incubation withBCTP at either dilution, influenza A virus infectivity of MDCK cells wascompletely abolished. There was no significant difference between theinteraction of BCTP with influenza A virus regardless of concentrationor time.

TABLE 22 BCTP treatment/dilution Time (min) 1:10 1:100 untreated 50.00^(a) 0.00 35.25 (+/−0.00)^(b) (+/−0.00) (+/−0.94) 10 0.00 0.25 39.25(+/−0.00) (+/−0.12) (+/−1.95) 15 0.00 0.25 31.50 (+/−0.00) (+/−0.12)(+/−1.05) 30 0.00 0.00 26.50 (+/−0.00) (+/−0.00) (+/−0.08)

Anti-influenza A efficacy of BCTP: Since TRITON X-100 detergent hasanti-viral activity (Maha and Igarashi, 1997; Portocala et al., 1976),it was investigated whether TRITON X-100 alone or combined withindividual BCTP components inhibits influenza A infectivity to the sameextent as BCTP. Influenza A virus was treated with: 1) BCTP, 2) thecombination of tri(n-butyl)phosphate, TRITON X-100, and soybean oil(TTO), 3) TRITON X-100 and soybean oil (TO), or 4) TRITON X-100 (T)alone. BCTP was significantly more effective against influenza A virusat 1:10 and 1:1100 dilutions (TRITON X-100 dilution of 1:500, and1:5000) than TRITON X1100 alone or mixed with the other componentstested (FIG. 23). At the dilution 1:1000, BCTP (TRITON X-100 dilution of1:50,000) was able to reduce influenza A infection of MDCK cells byapproximately 50% while TRITON X-100 alone at the same concentration wascompletely ineffective.

BCTP does not affect infectivity of non-enveloped virus: To investigatewhether BCTP may affect the infectivity of non-enveloped virus,genetically engineered adenovirus containing LacZ gene was used,encoding β-galactosidase. This adenovirus construct was deficient in thetransforming gene and therefore can replicate and transform onlypermissive cells containing the transforming gene of adenovirus 5. The293 cells, which constitutively express transforming gene, were employedto promote adenovirus replication and production of β-galactosidaseenzyme. As shown in FIG. 24, BCTP treatment did not affect the abilityof adenovirus to replicate and express β-galactosidase activity in 293cells. Both BCTP treated and untreated adenovirus produced approximately0.11 units of β-galactosidase enzyme.

Action of BCTP on enveloped virus: Since BCTP only altered theinfectivity of enveloped viruses, the action of this nanoemulsion onenveloped virus integrity was further investigated using electronmicroscopy. As shown in FIG. 25D, after a 60 min incubation with 1:100dilution of BCTP, the structure of adenovirus is unchanged. A fewrecognizable influenza A virions were located after 15 min incubationwith BCTP (FIG. 25B), however, no recognizable influenza A virions werefound after 1 h incubation. BCTP's efficacy against influenza A virusand its minimal toxicity to mucous membranes demonstrates its potentialas an effective disinfectant and agent for prevention of diseasesresulting from infection with enveloped viruses.

Example 13 Temperature and EDTA Effects on W205EC Treatment of S.typhimurium

FIGS. 31 and 32 show the treatment of Salmonellae with differentemulsions of the present invention with the addition of 0.1% EDTA. TheEDTA improved the bactericidal activity of the emulsion at both 40° C.(FIG. 32) and 50° C. (FIG. 33). The emulsions were tested at 10.0%,1.0%, and 0.1% dilutions.

Example 14 Antimicrobial Properties of X8PC and W₂₀5EC

As described above, the emulsion X8PC is composed of about 8 vol. % ofTRITON X-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol. % ofsoybean oil, and about 19 vol. % of DiH₂O and the emulsion W₂₀5EC iscomposed of from about 5 vol. % of TWEEN 20, from about 8 vol. % ofethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g.,soybean oil), and about 22 vol. % of DiH₂O. X8PC and W₂₀5EC were testedfor their ability to reduce the growth of a number of microorganismsunder various conditions. FIG. 35 shows the log reduction ofMycobacteria fortuitum by X8PC at 10%, 1% and 0.1% dilutions at roomtemperature and 37° C.

A 2% emulsion of W₂₀5EC (with and without 1%, 2%, and 3% Natrosol) eachshowed an approximately 2 log reduction in E. coli for both dry and wetbacteria after a 15 minute incubation at room temperature. A 2% emulsionof W₂₀5EC (with and without 1%, 2%, and 3% Natrosol) each showed anapproximately 4 log reduction in S. aureus for both dry and wet bacteriaafter a 15 minute incubation at room temperature. A 2% emulsion ofW₂₀5EC (with and without 1%, 2%, and 3% Natrosol) each showed anapproximately 3 log reduction in N. gonorrhoeae for wet bacteria after a15 minute incubation at room temperature.

A rubber surface experiment was conducted to test the bactericidalactivity of 1% W₂₀5EC at multiple temperatures and diluted in differenttypes of water. A one foot surface was smeared with 20 g of beltscrapings. S. typhimurium was manually sprayed onto the surface andallowed to dry for 20 minutes. The treatment was applied in three oneminute intervals with a one minute time pause between each interval. Aten minute incubation period at room temperature was allowed. Theresults are shown in FIG. 36. The data demonstrate that W₂₀5EC iseffective using diH₂O, distilled water, and tap water at eachtemperature tested.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inrelevant fields are intended to be within the scope of the followingclaims.

1.-58. (canceled)
 59. A pharmaceutical composition consisting of: anoil-in-water nanoemulsion, or a dilution thereof, wherein saidnanoemulsion consists of: a) about 5% by volume surfactant, wherein saidsurfactant is selected from the group consisting ofpolyoxyethylenesorbitan monolaurate and polyoxyethylenesorbitanmonooleate; b) about 8% by volume ethanol; c) about 64% by volume oil;d) about 1% by volume cetylpyridinium chloride; e) distilled water; andf) an interaction enhancer. 60-65. (canceled)
 66. The pharmaceuticalcomposition of claim 59, wherein said distilled water is sterile,pyrogen-free distilled water.
 67. The pharmaceutical composition ofclaim 59, wherein said interaction enhancer isethylenediaminetetraacetic acid
 68. The pharmaceutical composition ofclaim 59, further comprising an antimicrobial agent other than saidoil-in-water nanoemulsion.
 69. The pharmaceutical composition of claim68, wherein said antimicrobial agent other than said oil-in-waternanoemulsion is acyclovir.
 70. The pharmaceutical composition of claim59, wherein said oil-in-water nanoemulsion has a mean particle size of0.2-0.8 microns.
 71. The pharmaceutical composition of claim 59, whereinsaid oil-in-water nanoemulsion has a mean particle size less than 0.5microns.
 72. The pharmaceutical composition of claim 59, wherein saidoil-in-water nanoemulsion kills or disables Gram negative bacteria, Grampositive bacteria, viruses, spores, and fungi when in contact with saidGram negative bacteria, Gram positive bacteria, viruses, spores, andfungi.
 73. (canceled)